Aminobenzimidazole derivatives, treatments, and methods of inhibiting histone deacetylase

ABSTRACT

The present invention is directed to novel aminobenzimidazole derivatives. The present invention is also directed to methods of treating a histone deacetylase (HDAC)-associated disease or inhibiting the histone deacetylating activity of a HDAC isoform in a cell or with one or more of the aminobenzimidazole derivatives.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/554,728, filed Sep. 6, 2017, the contents of which areincorporated herein by reference in their entirety.

TECHNICAL FIELD

The disclosure relates to aminobenzimidazole compounds, pharmaceuticalcompositions, use of the compounds to inhibit histone deacetylase (HDAC)and treat HDAC-associated diseases.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of death in the United States.Despite breakthroughs that have led to decreased mortality, many cancersremain refractory to treatment. Also, many cancers often developresistance to current chemotherapies over time. The typical treatmentssuch as chemotherapy, radiotherapy, and surgery also cause a broadspectrum of undesirable side effects. Thus, there is a need for novelcompounds and methods of treating cancer and other histonedeacetylase-associated diseases.

Histone deacetylases (HDACs) are a family of enzymes that deacetylatehistones and non-histone proteins known to modulate gene transcription.HDACs have been associated with proliferation and differentiation ofvarious cell types as well as pathogenesis of diseases including cancer,interstitial fibrosis, autoimmune and inflammatory diseases, andmetabolic disorders.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to a compound of formula (I):

In some embodiments, X is selected from the group consisting of: H,halo, —C₁-C₆ alkyl, aryl, —C₃-C₇ cycloalkyl, and -3- to 10-memberedheterocycle, any of which is unsubstituted, or substituted with one ormore of: -halo, —C₁-C₆ alkyl, —O—(C₁-C₆ alkyl), —OH, —CN, —COOR′,—OC(O)R′, NHR′, N(R′)₂, —NHC(O)R′, and —C(O)NHR′, wherein R′ is —H or—C₁-C₆ alkyl; A is selected from the group consisting of: a bond, —C₁-C₆alkyl, and —C₃-C₇ cycloalkyl, any of which is unsubstituted, orsubstituted with one or more of: -halo, —C₁-C₆ alkyl, —O—(C₁-C₆ alkyl),—OH, —CN, —COOR′, —OC(O)R′, NHR′, N(R′)₂, —NHC(O)R′, and —C(O)NHR′,wherein R′ is —H or —C₁-C₆ alkyl; Y is selected from the groupconsisting of: H, —C₁-C₆ alkyl, —C₃-C₇ cycloalkyl, aryl, and -3- to10-membered heterocycle, any of which is unsubstituted, or substitutedwith one or more of: -halo, —C₁-C₆ alkyl, —O—(C₁-C₆ alkyl), —OH, —CN,—COOR′, —OC(O)R′, NHR′, N(R′)₂, —NHC(O)R′, and —C(O)NHR′, wherein R′ is—H or —C₁-C₆ alkyl; and Q is selected from the group consisting of: —H,-halo, —C₁-C₆ alkyl, —O—(C₁-C₆ alkyl), —OH, —CN, —COOR′, —OC(O)R′, NHR′,N(R′)₂, —NHC(O)R′, and —C(O)NHR′, wherein R′ is —H or —C₁-C₆ alkyl.

In other particular embodiments, X is H or halo; A is selected from thegroup consisting of: a bond, —C₁-C₆ alkyl, and —C₃-C₇ cycloalkyl, any ofwhich is unsubstituted, or substituted with one or more of: -halo,—C₁-C₆ alkyl, —O—(C₁-C₆ alkyl), —OH, —CN, —COOR′, —OC(O)R′, NHR′,N(R′)₂, —NHC(O)R′, and —C(O)NHR′, wherein R′ is —H or —C₁-C₆ alkyl; Y isselected from the group consisting of: H, —C₁-C₆ alkyl, —C₃-C₇cycloalkyl, aryl, and -3- to 10-membered heterocycle, any of which isunsubstituted, or substituted with one or more of: -halo, —C₁-C₆ alkyl,—O—(C₁-C₆ alkyl), —OH, —CN, —COOR′, —OC(O)R′, NHR′, N(R′)₂, —NHC(O)R′,and —C(O)NHR′, wherein R′ is —H or —C₁-C₆ alkyl; and Q is H.

In yet other embodiments, X is H or halo; A is —C₁-C₆ alkyl; Y isselected from the group consisting of: H, —C₁-C₆ alkyl, —C₃-C₇cycloalkyl, aryl, and -3- to 10-membered heterocycle, any of which isunsubstituted, or substituted with one or more of: -halo, —C₁-C₆ alkyl,—O—(C₁-C₆ alkyl), —OH, —CN, —COOR′, —OC(O)R′, NHR′, N(R′)₂, —NHC(O)R′,and —C(O)NHR′, wherein R′ is —H or —C₁-C₆ alkyl; and Q is H.

In further embodiments, X is H or halo; A is —C₁-C₆ alkyl; Y is —C₃-C₇cycloalkyl, unsubstituted, or substituted with one or more of: -halo and-3- to 10-membered heterocycle; and and Q is H.

In yet further embodiments, X is H or halo; A is —C₁-C₃ alkyl; Y isselected from the group consisting of: H, —C₁-C₆ alkyl, —C₃-C₇cycloalkyl, aryl, and -3- to 10-membered heterocycle, any of which isunsubstituted, or substituted with one or more of: -halo, —C₁-C₆ alkyl,—O—(C₁-C₆ alkyl), —OH, —CN, —COOR′, —OC(O)R′, NHR′, N(R′)₂, —NHC(O)R′,and —C(O)NHR′, wherein R′ is —H or —C₁-C₆ alkyl; and Q is H.

In certain embodiments, X is H or halo; A is —C₁-C₃ alkyl; Y is —C₃-C₇cycloalkyl, unsubstituted, or substituted with one or more of: -halo and-3- to 10-membered heterocycle; and and Q is H.

Non-limiting examples of the compound of formula (I) include:

4-((1-(cyclohexylmethyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #1)

4-((1-cyclohexyl-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide (ID#2)

4-((1-cycloheptyl-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide (ID#3)

4-((1-(1-fluorocyclohexyl)methyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #4)

4-((1-(cyclopentylmethyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #5)

4-((1-(2-cyclopentylethyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #6)

4-((1-(4,4-difluorocyclohexyl)methyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #7)

4-((1-(4,4-difluorocyclohexyl)methyl)-5-fluoro-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #8)

N-hydroxy-4-((1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide(ID #9)

and

4-((5-fluoro-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #10)

In other more specific aspects of the invention, the compound of formula(I) is4-((1-(cyclohexylmethyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #1)

The invention is also directed to a method of inhibiting the histonedeacetylating activity of a histone deacetylase (HDAC) isoform in acell. The method typically comprises contacting the cell with thecompound of formula (I), wherein the compound of formula (I) inhibitsthe histone deacetylating activity of the HDAC isoform in the cell. Inspecific embodiments, the method is used to treat a disease associatedwith a histone deacetylase (HDAC) isoform.

In certain embodiments, the invention is directed to a method ofinhibiting an HDAC isoform in one or more various cells, for example, acancer cell, a neuronal cell, a cell of the immune system, a cell of thecirculatory system, or combinations thereof. Non-limiting examples ofcancer cells include acute lymphocytic leukemia (ALL) cell, acutemyeloid leukemia (AML) cell, acute promyelocytic leukemia (APL) cell,breast cancer cell, chronic myeloid leukemia (CML) cell, colon cancercell, diffuse large B-cell lymphoma (DLBCL) cell, gastrointestinalstromal tumor (GIST) cell, glioblastoma (GBM) cell, hepatocellularcarcinoma cell, Hodgkin lymphoma cell, leukemia cell, lung cancer cell,multiple myeloma cell, non-Hodgkin's lymphoma cell, non-small cell lungcancer (NSCLC) cell, neuroblastoma cell, ovarian cancer cell, pancreaticductal adenocarcinoma cell, peripheral T-cell lymphoma cell, prostatecancer cell, uterine cancer cell, Waldenstrom myeloma cell, andcombinations thereof.

Non-limiting examples of HDAC isoforms include HDAC1, HDAC3, HDAC6, andHDAC10. In one embodiment, the compound of formula (I) inhibits thehistone deacetylating activity of the HDAC isoform with a half maximalinhibitory concentration (IC₅₀) of 0.001-10 μM, or in a more specificembodiment, 0.005-4 μM. In one aspect, the compound of formula (I)inhibits the histone deacetylating activity of the HDAC isoform by least30%, or in a more specific aspect, 30-90%. In some aspects, the compoundof formula (I) inhibits the histone deacetylating activity of the HDACisoform, thereby inhibits cell proliferation, induces cell death, orboth.

The invention is further directed to a method of treating a subjecthaving a disease by inhibiting a histone deacetylase (HDAC) isoform. Themethod typically comprises administering to the subject the compound offormula (I), wherein the compound of formula (I) inhibits the histonedeacetylating activity of the HDAC isoform. In some aspects, the subjectis a human.

Non-limiting examples of diseases include: cell-proliferative diseases(e.g., cancer), autoimmune disorders, inflammatory disorders,neurodegenerative diseases, and combinations thereof. In someembodiments, the cell-proliferative disease is a cancer, for example,acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), acutepromyelocytic leukemia (APL), breast cancer, chronic myeloid leukemia(CML), colon cancer, diffuse large B-cell lymphoma (DLBCL),gastrointestinal stromal tumor (GIST), glioblastoma (GBM),hepatocellular carcinoma, Hodgkin's lymphoma, leukemia, lung cancer,multiple myeloma, non-Hodgkin's lymphoma, non-small cell lung cancer(NSCLC), neuroblastoma, ovarian cancer, pancreatic ductaladenocarcinoma, peripheral T-cell lymphoma, prostate cancer, uterinecancer, and Waldenstrom myeloma. Non-limiting examples of autoimmune orinflammatory disorders include: airway hyperresponsiveness, Crohn'sdisease, inflammatory bowel disease, multiple sclerosis, psoriasis,rheumatoid arthritis, systemic lupus erythematosus, type 1 diabetes, andulcerative colitis. Non-limiting examples of neurodegenerative disordersinclude: Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS),cerebral ischemia, Huntington's disease (HD), Parkinson's disease (PD),and spinal muscular atrophy.

In some aspects, the compound of formula (I) inhibits the histonedeacetylating activity of the HDAC isoform, thereby inhibitsproliferation, induces death, or both of certain cells. Nonlimitingexamples of such cells include: cancer cells, neuronal cells, cells ofthe immune system, cells of the circulatory system, or combinationsthereof. Non-limiting examples of cancer cells include: acutelymphocytic leukemia (ALL) cell, acute myeloid leukemia (AML) cell,acute promyelocytic leukemia (APL) cell, breast cancer cell, chronicmyeloid leukemia (CML) cell, colon cancer cell, diffuse large B-celllymphoma (DLBCL) cell, gastrointestinal stromal tumor (GIST) cell,glioblastoma (GBM) cell, hepatocellular carcinoma cell, Hodgkin lymphomacell, leukemia cell, lung cancer cell, multiple myeloma cell,non-Hodgkin's lymphoma cell, non-small cell lung cancer (NSCLC) cell,neuroblastoma cell, ovarian cancer cell, pancreatic ductaladenocarcinoma cell, peripheral T-cell lymphoma cell, prostate cancercell, uterine cancer cell, Waldenstrom myeloma cell, and combinationsthereof.

In some aspects, the compound of formula (I) is in a pharmaceuticallyacceptable carrier. In one embodiment, the composition is administeredsystemically. In another embodiment, the composition is administeredlocally. In yet another embodiment, the composition is administeredorally or intravenously. In one embodiment, the composition isadministered about every 6, 8, 12, 16, 20, or 24 hours. In oneembodiment, the compound of formula (I) is administered at 10-400 mg/kg,or in a more specific embodiment, at 40-250 mg/kg.

Non-limiting examples of HDAC isoforms include: HDAC1, HDAC3, HDAC6, andHDAC10. In one embodiment, the compound of formula (I) inhibits thehistone deacetylating activity of the HDAC isoform with a half maximalinhibitory concentration (IC₅₀) of 0.001-10 μM, or in a more specificembodiment, 0.005-4 μM. In one aspect, the compound of formula (I)inhibits the histone deacetylating activity of the HDAC isoform by least30%, or in a more specific aspect, 30-90%.

In some aspects, the subject is further administered a chemotherapydrug. Non-limiting examples of chemotherapy drugs include: pomalidomide,dexamethasone, and a combination thereof.

The invention is further directed to a method of treating a histonedeacetylase (HDAC)-associated disease in a subject. The method typicallycomprises administering to the subject the compound of formula (I). Insome embodiments, the compound of formula (I) is administered in atherapeutically effective amount. In some aspects, the subject is ahuman.

Non-limiting examples of HDAC-associated diseases include:cell-proliferative diseases (e.g., cancer), autoimmune disorders,inflammatory disorders, neurodegenerative diseases, and combinationsthereof. In some embodiments, the cell-proliferative disease is acancer, for example, acute lymphocytic leukemia (ALL), acute myeloidleukemia (AML), acute promyelocytic leukemia (APL), breast cancer,chronic myeloid leukemia (CML), colon cancer, diffuse large B-celllymphoma (DLBCL), gastrointestinal stromal tumor (GIST), glioblastoma(GBM), hepatocellular carcinoma, Hodgkin's lymphoma, leukemia, lungcancer, multiple myeloma, non-Hodgkin's lymphoma, non-small cell lungcancer (NSCLC), neuroblastoma, ovarian cancer, pancreatic ductaladenocarcinoma, peripheral T-cell lymphoma, prostate cancer, uterinecancer, and Waldenstrom myeloma. Non-limiting examples of autoimmune orinflammatory disorders include: airway hyperresponsiveness, Crohn'sdisease, inflammatory bowel disease, multiple sclerosis, psoriasis,rheumatoid arthritis, systemic lupus erythematosus, type 1 diabetes, andulcerative colitis. Non-limiting examples of neurodegenerative disordersinclude: Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS),cerebral ischemia, Huntington's disease (HD), Parkinson's disease (PD),and spinal muscular atrophy.

In some aspects, the compound of formula (I) is in a pharmaceuticallyacceptable carrier. In one embodiment, the composition is administeredsystemically. In another embodiment, the composition is administeredlocally. In yet another embodiment, the composition is administeredorally or intravenously. In one embodiment, the composition isadministered about every 6, 8, 12, 16, 20, or 24 hours. In oneembodiment, the compound of formula (I) is administered at 10-400 mg/kg,or in a more specific embodiment, at 40-250 mg/kg. In some aspects, thesubject is further administered a chemotherapy drug. Non-limitingexamples of chemotherapy drugs include: pomalidomide, dexamethasone, anda combination thereof.

In some aspects, the disease is associated with HDAC1, HDAC3, HDAC6,HDAC10, or combinations thereof. In one embodiment, the compound offormula (I) inhibits the histone deacetylating activity of the HDACisoform associated with the disease with a half maximal inhibitoryconcentration (IC₅₀) of 0.001-10 μM, or in a more specific embodiment,0.005-4 μM. In one aspect, the compound of formula (I) inhibits thehistone deacetylating activity of the HDAC isoform associated with thedisease by least 30%, or in a more specific aspect, 30-90%.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the effect of treatment with Compound ID #1, Compound ID#2, or Compound ID #3 on tumor volume in a human myeloma tumor xenograftmodel.

FIG. 2 depicts the effect of treatment with Compound ID #1, Compound ID#2, or Compound ID #3 in combination with dexamethasone and pomalidomideon tumor volume in a human myeloma tumor xenograft model.

DETAILED DESCRIPTION OF THE INVENTION

In the following description, and for the purposes of explanation,numerous specific details are set forth in order to provide a thoroughunderstanding of the various aspects of the invention. It will beunderstood, however, by those skilled in the relevant arts, that thepresent invention may be practiced without these specific details. Inother instances, known structures and devices are shown or discussedmore generally in order to avoid obscuring the invention. In many cases,a description of the operation is sufficient to enable one to implementthe various forms of the invention. It should be noted that there aremany different and alternative configurations, devices, compositions,and technologies to which the disclosed invention may be applied. Thefull scope of the inventions is not limited to the examples that aredescribed below.

Compounds of Formula (I)

Herein the inventors disclose a compound of formula (I):

A compound of formula (I):

wherein:

-   -   X is selected from the group consisting of: H, halo, —C₁-C₆        alkyl, aryl, —C₃-C₇ cycloalkyl, and -3- to 10-membered        heterocycle, any of which is unsubstituted, or substituted with        one or more of: -halo, —C₁-C₆ alkyl, —O—(C₁-C₆ alkyl), —OH, —CN,        —COOR′, —OC(O)R′, NHR′, N(R′)₂, —NHC(O)R′, and —C(O)NHR′,        wherein R′ is —H or —C₁-C₆ alkyl;    -   A is selected from the group consisting of: a bond, —C₁-C₆        alkyl, and —C₃-C₇ cycloalkyl, any of which is unsubstituted, or        substituted with one or more of: -halo, —C₁-C₆ alkyl, —O—(C₁-C₆        alkyl), —OH, —CN, —COOR′, —OC(O)R′, NHR′, N(R′)₂, —NHC(O)R′, and        —C(O)NHR′, wherein R′ is —H or —C₁-C₆ alkyl;    -   Y is selected from the group consisting of: H, —C₁-C₆ alkyl,        —C₃-C₇ cycloalkyl, aryl, and -3- to 10-membered heterocycle, any        of which is unsubstituted, or substituted with one or more of:        -halo, —C₁-C₆ alkyl, —O—(C₁-C₆ alkyl), —OH, —CN, —COOR′,        —OC(O)R′, NHR′, N(R′)₂, —NHC(O)R′, and —C(O)NHR′, wherein R′ is        —H or —C₁-C₆ alkyl; and    -   Q is selected from the group consisting of: —H, -halo, —C₁-C₆        alkyl, —O—(C₁-C₆ alkyl), —OH, —CN, —COOR′, —OC(O)R′, NHR′,        N(R′)₂, —NHC(O)R′, and —C(O)NHR′, wherein R′ is —H or —C₁-C₆        alkyl.

In some embodiments, X is H or halo. In other embodiments, Q is H. Inyet other embodiments, A is —C₁-C₆ alkyl. In further embodiments, A is—C₁-C₃ alkyl. In yet further embodiments, Y is —C₃-C₇ cycloalkyl,unsubstituted, or substituted with one or more of: -halo and -3- to10-membered heterocycle.

In some aspects, X is H or halo; A is selected from the group consistingof: a bond, —C₁-C₆ alkyl, and —C₃-C₇ cycloalkyl, any of which isunsubstituted, or substituted with one or more of: -halo, —C₁-C₆ alkyl,—O—(C₁-C₆ alkyl), —OH, —CN, —COOR′, —OC(O)R′, NHR′, N(R′)₂, —NHC(O)R′,and —C(O)NHR′, wherein R′ is —H or —C₁-C₆ alkyl; Y is selected from thegroup consisting of: H, —C₁-C₆ alkyl, —C₃-C₇ cycloalkyl, aryl, and -3-to 10-membered heterocycle, any of which is unsubstituted, orsubstituted with one or more of: -halo, —C₁-C₆ alkyl, —O—(C₁-C₆ alkyl),—OH, —CN, —COOR′, —OC(O)R′, NHR′, N(R′)₂, —NHC(O)R′, and —C(O)NHR′,wherein R′ is —H or —C₁-C₆ alkyl; and Q is —H in the compound of formula(I).

In other aspects, X is H or halo; A is —C₁-C₆ alkyl; Y is selected fromthe group consisting of: H, —C₁-C₆ alkyl, —C₃-C₇ cycloalkyl, aryl, and-3- to 10-membered heterocycle, any of which is unsubstituted, orsubstituted with one or more of: -halo, —C₁-C₆ alkyl, —O—(C₁-C₆ alkyl),—OH, —CN, —COOR′, —OC(O)R′, NHR′, N(R′)₂, —NHC(O)R′, and —C(O)NHR′,wherein R′ is —H or —C₁-C₆ alkyl; and Q is —H in the compound of formula(I).

In yet other aspects, X is H or halo; A is —C₁-C₃ alkyl; Y is selectedfrom the group consisting of: H, —C₁-C₆ alkyl, —C₃-C₇ cycloalkyl, aryl,and -3- to 10-membered heterocycle, any of which is unsubstituted, orsubstituted with one or more of: -halo, —C₁-C₆ alkyl, —O—(C₁-C₆ alkyl),—OH, —CN, —COOR′, —OC(O)R′, NHR′, N(R′)₂, —NHC(O)R′, and —C(O)NHR′,wherein R′ is —H or —C₁-C₆ alkyl; and Q is —H in the compound of formula(I).

In further aspects, X is H or halo; A is selected from the groupconsisting of: a bond, —C₁-C₆ alkyl, and —C₃-C₇ cycloalkyl, any of whichis unsubstituted, or substituted with one or more of: -halo, —C₁-C₆alkyl, —O—(C₁-C₆ alkyl), —OH, —CN, —COOR′, —OC(O)R′, NHR′, N(R′)₂,—NHC(O)R′, and —C(O)NHR′, wherein R′ is —H or —C₁-C₆ alkyl; Y is —C₃-C₇cycloalkyl or —C₃-C₇ cycloalkyl substituted with one or more of: -haloand -3- to 10-membered heterocycle; and Q is —H in the compound offormula (I).

In yet further aspects, X is H or halo; A is —C₁-C₆ alkyl; Y is —C₃-C₇cycloalkyl or —C₃-C₇ cycloalkyl substituted with one or more of: -haloand -3- to 10-membered heterocycle; and Q is —H in the compound offormula (I).

In certain aspects, X is H or halo; A is —C₁-C₃ alkyl; Y is —C₃-C₇cycloalkyl or —C₃-C₇ cycloalkyl substituted with one or more of: -haloand -3- to 10-membered heterocycle; and Q is —H in the compound offormula (I).

Non-limiting examples of compound of formula (I) include:

4-((1-(cyclohexylmethyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #1)

4-((1-cyclohexyl-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide (ID#2)

4-((1-cycloheptyl-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide (ID#3)

4-((1-(1-fluorocyclohexyl)methyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #4)

4-((1-(cyclopentylmethyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #5)

4-((1-(2-cyclopentylethyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #6)

4-((1-((4,4-difluorocyclohexyl)methyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #7)

4-((1-(4,4-difluorocyclohexyl)methyl)-5-fluoro-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #8)

N-hydroxy-4-((1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide(ID #9)

and

4-((5-fluoro-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #10)

In some embodiments, the compound is selected from the group consistingof:4-((1-(cyclohexylmethyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #1),4-((1-(1-fluorocyclohexyl)methyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #4),4-((1-(cyclopentylmethyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #5),4-((1-(2-cyclopentylethyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #6),4-((1-((4,4-difluorocyclohexyl)methyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #7),4-((1-(4,4-difluorocyclohexyl)methyl)-5-fluoro-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #8),N-hydroxy-4-((1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide(ID #9), and4-((5-fluoro-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #10).

In other embodiments, the compound of formula (I) is4-((1-(cyclohexylmethyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #1)

A —C₁-C₆ alkyl includes any straight or branched, saturated orunsaturated, substituted or unsubstituted hydrocarbon comprised ofbetween one and six carbon atoms. Non-limiting examples of —C₁-C₆ alkylinclude: methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl,pentyl, isopentyl, neopentyl, hexyl, isohexyl, neohexyl, ethylenyl,propylenyl, 1-butenyl, 2-butenyl, 1-pentenyl, 2-pentenyl, 1-hexenyl,2-hexenyl, 3-hexenyl, acetylenyl, pentynyl, 1-butynyl, 2-butynyl,1-pentynyl, 2-pentynyl, 1-hexynyl, 2-hexynyl, and 3-hexynyl, etc.Non-limiting examples of substituting groups include: halo, —C₁-C₆alkyl, —O—(C₁-C₆ alkyl), —OH, —CN, —COOR′, —OC(O)R′, —NHR′, and N(R′)₂,—NHC(O)R′, and —C(O)NHR′, wherein R′ is —H or —C₁-C₆ alkyl.

An aryl includes any unsubstituted or substituted phenyl or napthyl.Non-limiting examples of substituting groups include: halo, —C₁-C₆alkyl, —O—(C₁-C₆ alkyl), —OH, —CN, —COOR′, —OC(O)R′, NHR′, N(R′)₂,—NHC(O), R′, and —C(O)NEtR′, wherein R′ is —H or —C₁-C₆ alkyl.

A C₃-C₇ cycloalkyl includes any 3-, 4-, 5-, 6-, or 7-memberedsubstituted or unsubstituted non-aromatic carbocyclic ring. Non-limitingexamples of C₃-C₇ cycloalkyl include: cyclopropyl, cyclobutyl,cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, cycloheptyl,cycloheptanyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl,1,3-cycloheptadienyl, and 1,3,5-cycloheptatrienyl. Non-limiting examplesof substituting groups include: -halo, —C₁-C₆ alkyl, —O—(C₁-C₆ alkyl),—OH, —CN, —COOR′, —OC(O)R′, NHR′, N(R′)₂, —NHC(O)R′, and —C(O)NHR′,wherein R′ is —H or unsubstituted —C₁-C₆ alkyl.

A halo includes any halogen, non-limiting examples include: —F, —Cl,—Br, and —I.

A heterocycle is an optionally substituted, saturated, unsaturated, oraromatic cyclic moiety, wherein the cyclic moiety is interrupted by atleast one heteroatom selected from the group consisting of: oxygen (O),sulfur (S), and nitrogen (N). A heterocycle is a monocyclic orpolycyclic ring. A suitable substituents is selected from the groupconsisting of: (1) halogen, halogenated —C₁-C₆ alkyl, halogenated —C₁-C₆alkoxy, amino, amidino, amido, azido, cyano, guanidino, hydroxyl, nitro,nitroso, and urea; (2) OS(O)₂R, OS(O)₂OR, S(O)₂OR S(O)₀₋₂R, and C(O)OR,wherein R is H, C₁-C₆ alkyl, aryl or 3- to 10-membered heterocycle; (3)OP(O)OR₁OR₂, P(O)OR₁OR₂, SO₂NR₁R₂, NR₁SO₂R₂, C(R₁)NR₂, and C(R₁)NOR₂,wherein R₁ and R₂ is independently H, C₁-C₆ alkyl, aryl, or 3- to10-membered heterocycle; and (4) NR₁C(O)R₂, NR₁C(O)OR₂, NR₃C(O)NR₂R₁,C(O)NR₁R₂, and OC(O)NR₁R₂, wherein R₁, R₂ and R₃ are each independentlyselected from the group consisting of: H, C₁-C₆ alkyl, aryl, and 3- to10-membered heterocycle, or R₁ and R₂ are taken together with the atomsto which they are attached to form a 3- to 10-membered heterocycle.

Non-limiting examples of the substituent of the heterocycle include:halogen (Br, Cl, I, or F), cyano, nitro, oxo, amino, C₁₋₄ alkyl (e.g.,CH₃, C₂H₅, or isopropyl), C₁₋₄ alkoxy (e.g., OCH₃ or OC₂H₅), halogenatedC₁₋₄ alkyl (e.g., CF₃ or CHF₂), halogenated C₁₋₄ alkoxy (e.g., OCF₃ orOC₂F₅), COOH, COO—C₁₋₄ alkyl, CO—C₁₋₄ alkyl, C₁₋₄ alkyl —S— (e.g., CH₃Sor C₂H₅S), halogenated C₁₋₄ alkyl —S— (e.g., CF₃S or C₂F₅S), benzyloxy,and pyrazolyl.

Non-limiting examples of heterocycles include: azepinyl, aziridinyl,azetyl, azetidinyl, diazepinyl, dithiadiazinyl, dioxazepinyl,dioxolanyl, dithiazolyl, furanyl, isooxazolyl, isothiazolyl, imidazolyl,morpholinyl, morpholino, oxetanyl, oxadiazolyl, oxiranyl, oxazinyl,oxazolyl, piperazinyl, pyrazinyl, pyridazinyl, pyrimidinyl, piperidyl,piperidino, pyridyl, pyranyl, pyrazolyl, pyrrolyl, pyrrolidinyl,thiatriazolyl, tetrazolyl, thiadiazolyl, triazolyl, thiazolyl, thienyl,tetrazinyl, thiadiazinyl, triazinyl, thiazinyl, thiopyranylfuroisoxazolyl, imidazothiazolyl, thienoisothiazolyl, thienothiazolyl,imidazopyrazolyl, cyclopentapyrazolyl, pyrrolopyrrolyl, thienothienyl,thiadiazolopyrimidinyl, thiazolothiazinyl, thiazolopyrimidinyl,thiazolopyridinyl, oxazolopyrimidinyl, oxazolopyridyl, benzoxazolyl,benzisothiazolyl, benzothiazolyl, imidazopyrazinyl, purinyl,pyrazolopyrimidinyl, imidazopyridinyl, benzimidazolyl, indazolyl,benzoxathiolyl, benzodioxolyl, benzodithiolyl, indolizinyl, indolinyl,isoindolinyl, furopyrimidinyl, furopyridyl, benzofuranyl,isobenzofuranyl, thienopyrimidinyl, thienσpyridyl, benzothienyl,cyclopentaoxazinyl, cyclopentafuranyl, benzoxazinyl, benzothiazinyl,quinazolinyl, naphthyridinyl, quinolinyl, isoquinolinyl, benzopyranyl,pyridopyridazinyl, and pyridopyrimidinyl.

In some embodiments, the disclosed compound and its intermediates existin different tautomeric forms. Tautomers include any structural isomersof different energies that have a low energy barrier to interconversion.One example is proton tautomers (prototropic tautomers). In thisexample, the interconversions occur via the migration of a proton.Non-limiting examples of prototropic tautomers include keto-enol andimine-enamine isomerizations. In other embodiments (illustratedgraphically below), proton migrates between the 1-position and3-position nitrogen atoms of the benzimidazole ring. As a result,Formulas Ia and Ib are tautomeric forms of each other:

The invention encompasses any other physiochemical or stereochemicalform that the disclosed compound may assume. Such form includesdiastereomers, racemates, isolated enantiomers, hydrated forms, solvatedforms, or any other known or yet to be disclosed crystalline,polymorphic crystalline, or amorphous form. Amorphous forms lack adistinguishable crystal lattice and therefore lack an orderlyarrangement of structural units. Many pharmaceutical compounds haveamorphous forms. Methods of generating such chemical forms will be wellknown by one with skill in the art.

In some aspects of the invention the disclosed compound is in the formof a pharmaceutically acceptable salt. Pharmaceutically acceptable saltsinclude any salt derived from an organic or inorganic acid. Non-limitingexamples of such salts include: salts of hydrobromic acid, hydrochloricacid, nitric acid, phosphoric acid, and sulphuric acid. Non-limitingexamples of the organic acid addition salt include: salts of aceticacid, benzenesulphonic acid, benzoic acid, camphorsulphonic acid, citricacid, 2-(4-chlorophenoxy)-2-methylpropionic acid, 1,2-ethanedisulphonicacid, ethanesulphonic acid, ethylenediaminetetraacetic acid (EDTA),fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid,N-glycolylarsanilic acid, 4-hexylresorcinol, hippuric acid,2-(4-hydroxybenzoyl) benzoicacid, 1-hydroxy-2-naphthoicacid,3-hydroxy-2-naphthoic acid, 2-hydroxyethanesulphonic acid, lactobionicacid, n-dodecyl sulphuric acid, maleic acid, malic acid, mandelic acid,methanesulphonic acid, methyl sulpuric acid, mucic acid,2-naphthalenesulphonic acid, pamoic acid, pantothenic acid, phosphanilicacid ((4-aminophenyl) phosphonic acid), picric acid, salicylic acid,stearic acid, succinic acid, tannic acid, tartaric acid, terephthalicacid, p-toluenesulphonic acid, 10-undecenoic acid, and any other acidnow known or yet to be disclosed. It will be appreciated by one skilledin the art that such pharmaceutically acceptable salts may be used inthe formulation of a pharmacological composition. Such salts may beprepared by reacting the disclosed compound with a suitable acid in amanner known by those skilled in the art.

The invention further encompasses aspects in which a protecting group isadded to the compound. One skilled in the art would recognize thatduring the synthesis of complex molecules, one group on the disclosedcompound may happen to interfere with an intended reaction that includesa second group on the compound. Temporarily masking or protecting thefirst group encourages the desired reaction. Protection involvesintroducing a protecting group to a group to be protected, carrying outthe desired reaction, and removing the protecting group Removal of theprotecting group may be referred to as deprotection. Examples ofcompounds to be protected in some syntheses include: hydroxy groups,amine groups, carbonyl groups, carboxyl groups, and thiols.

Many protective groups and reagents capable of introducing them intosynthetic processes have been and are continuing to be developed today.A protecting group may result from any chemical synthesis thatselectively attaches a group that is resistant to certain reagents tothe chemical group to be protected without significant effects on anyother chemical groups in the molecule, remains stable throughout thesynthesis, and may be removed through conditions that do not adverselyreact with the protected group, nor any other chemical group in themolecule. Multiple protecting groups may be added throughout a synthesisand one skilled in the art would be able to develop a strategy forspecific addition and removal of the protecting groups to and from thegroups to be protected.

Protecting groups, reagents that add those groups, preparations of thosereagents, protection and deprotection strategies under a variety ofconditions, including complex syntheses with mutually complementaryprotecting groups are all well known in the art. Nonlimiting examples ofall of these may be found in Green et al, Protective Groups in OrganicChemistry 2^(nd) Ed., (Wiley 1991), and Harrison et al, Compendium ofSynthetic Organic Methods, Vols. 1-8 (Wiley, 1971-1996) both of whichhereby incorporated by reference in its entirety.

Racemates, individual enantiomers, or diasteromers of the disclosedcompound may be prepared by specific synthesis or resolution through anymethod now known or yet to be disclosed. For example, the disclosedcompound may be resolved into it enantiomers by the formation ofdiasteromeric pairs through salt formation using an optically activeacid. Enantiomers are fractionally crystallized and the free baseregenerated. In another example, enantiomers may be separated bychromatography. Such chromatography may be any appropriate method nowknown or yet to be disclosed that is appropriate to separate enantiomerssuch as HPLC on a chiral column.

Pharmaceutical Compositions Comprising the Compound of Formula (I)

The invention further encompasses pharmaceutical compositions thatcomprise any one of the disclosed compounds of formula (I) as aningredient.

Such pharmaceutical compositions may take any physical form necessarydepending on a number of factors including the desired method ofadministration and the physicochemical and stereochemical form taken bythe disclosed compound or pharmaceutically acceptable salts of thecompound. Such physical forms include a solid, liquid, gas, sol, gel,aerosol, or any other physical form now known or yet to be disclosed.The concept of a pharmaceutical composition including the disclosedcompound also encompasses the disclosed compound or a pharmaceuticallyacceptable salt thereof without any other additive. The physical form ofthe invention may affect the route of administration and one skilled inthe art would know to choose a route of administration that takes intoconsideration both the physical form of the compound and the disorder tobe treated. Pharmaceutical compositions that include one of thedisclosed compounds may be prepared using methodology well known in thepharmaceutical art. A pharmaceutical composition that includes one ofthe disclosed compounds may include a second effective compound of adistinct chemical formula from the disclosed compound. This secondeffective compound may have the same or a similar molecular target asthe target or it may act upstream or downstream of the molecular targetof the disclosed compound with regard to one or more biochemicalpathways.

Pharmaceutical compositions including one of the disclosed compoundsinclude materials capable of modifying the physical form of a dosageunit. In one nonlimiting example, the composition includes a materialthat forms a coating that holds in the compound. Materials that may beused in such a coating include, for example, sugar, shellac, gelatin, orany other inert coating agent.

Pharmaceutical compositions including one of the disclosed compounds maybe prepared as a gas or aerosol. Aerosols encompass a variety of systemsincluding colloids and pressurized packages. Delivery of a compositionin this form may include propulsion of a pharmaceutical compositionincluding the disclosed compound through use of liquefied gas or othercompressed gas or by a suitable pump system. Aerosols may be deliveredin single-phase, bi-phasic, or tri-phasic systems.

In some aspects of the invention, the pharmaceutical composition one ofthe disclosed compounds is in the form of a solvate. Such solvates areproduced by the dissolution of the disclosed compound in apharmaceutically acceptable solvent. Pharmaceutically acceptablesolvents include any mixtures of more than one solvent. Such solventsmay include: pyridine, chloroform, propan-1-ol, ethyl oleate, ethyllactate, ethylene oxide, water, ethanol, and any other solvent thatdelivers a sufficient quantity of the disclosed compound to treat theaffliction without serious complications arising from the use of thesolvent in a majority of patients.

Pharmaceutical compositions that include one of the disclosed compoundsmay also include a pharmaceutically acceptable carrier. Carriers includeany substance that may be administered with the disclosed compound withthe intended purpose of facilitating, assisting, or helping theadministration or other delivery of the compound. Carriers include anyliquid, solid, semisolid, gel, aerosol or anything else that may becombined with the disclosed compound to aid in its administration.Examples include diluents, adjuvants, excipients, water, oils (includingpetroleum, animal, vegetable, or synthetic oils). Such carriers includeparticulates such as a tablet or powder, liquids such as an oral syrupor injectable liquid and inhalable aerosols. Further examples includesaline, gum acacia, gelatin, starch paste, talc, keratin, colloidalsilica, and urea. Such carriers may further include binders such asethyl cellulose, carboxymethylcellulose, microcrystalline cellulose, orgelatin; excipients such as starch, lactose or dextrins; disintegratingagents such as alginic acid, sodium alginate, Primogel, and corn starch;lubricants such as magnesium stearate or Sterotex; glidants such ascolloidal silicon dioxide; sweetening agents such as sucrose orsaccharin, a flavoring agent such as peppermint, methyl salicylate ororange flavoring, or coloring agents. Further examples of carriersinclude: polyethylene glycol, cyclodextrin, oils, or any other similarliquid carrier that may be formulated into a capsule. Still furtherexamples of carriers include: sterile diluents such as water forinjection, saline solution, physiological saline, Ringer's solution,isotonic sodium chloride, fixed oils such as synthetic mono ordigylcerides, polyethylene glycols, glycerin, cyclodextrin, propyleneglycol or other solvents; antibacterial agents such as benzyl alcohol ormethyl paraben; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose, thickening agents,lubricating agents, and coloring agents.

The pharmaceutical composition including one of the disclosed compoundsmay take any of a number of formulations depending on thephysicochemical form of the composition and the type of administration.Such forms include: solutions, suspensions, emulsions, tablets, pills,pellets, capsules, capsules including liquids, powders,sustained-release formulations, directed release formulations,lyophylates, suppositories, emulsions, aerosols, sprays, granules,powders, syrups, elixirs, or any other formulation now known or yet tobe disclosed. Additional examples of suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin,hereby incorporated by reference in its entirety.

Pharmaceutical compositions including one of the disclosed compounds maybe prepared in a form that facilitates topical or transdermaladministration. Such preparations may be in the form of a solution,emulsion, ointment, gel base, transdermal patch or iontophoresis device.Examples of bases used in such compositions include: petrolatum,lanolin, polyethylene glycols, beeswax, mineral oil, diluents such aswater and alcohol, and emulsifiers and stabilizers, thickening agents,or any other suitable base now known or yet to be disclosed.

HDAC and Associated Diseases

Histone acetyltransferases (HAT) impact gene expression by controllingthe coiling and uncoiling of DNA around histones. Histoneacetyltransferases accomplish this by acetylating lysine residues incore histones leading to less compact and more transcriptionally activechromatin. In contrast, histone deacetylases (HDAC) remove the acetylgroups from lysine residues, leading to a more condensed andtranscriptionally silenced chromatin. Reversible modification of theterminal tails of core histones constitutes the major epigeneticmechanism for remodeling of higher-order chromatin structure andcontrolling gene expression. HDAC inhibitors (HDI) block this action andcan result in hyperacetylation of histones, thereby affecting geneexpression. Thagalingam S., Cheng K H, Lee H J et al., Ann. N.Y. Acad.Sci. 983: 84-100, 2003; Marks P A. Richon V M, Rifkind R A, J. Natl.Cancer Inst. 92 (15) 1210-16, 2000; Dokmanovic M, Clarke C., Marks P A,Mol. Cancer Res. 5 (10) 981-989, 2007.

Histone deacetylase (HDAC) inhibitors are a new class of cytostaticagents that inhibit the proliferation of tumor cells in culture and invivo by inducing cell cycle arrest, differentiation and/or apoptosis.Acetylation and deacetylation play important roles in the modulation ofchromatin topology and the regulation of gene transcription. Histonedeacetylase inhibitors induce the accumulation of hyperacetylatednucleosome core histones in many regions of chromatin but affect theexpression of only a small subset of genes, leading to transcriptionalactivation of some genes, but repression of an equal or larger number ofother genes. Non-histone proteins such as transcription factors are alsotargets for acetylation with varying functional effects. Acetylationenhances the activity of some transcription factors such as the tumorsuppressor p53 and the erythroid differentiation factor GATA-1 but mayrepress the transcriptional activity of others including T cell factorand the co-activator ACTR. Recent studies have shown that the estrogenreceptor alpha (ERalpha) can be hyperacetylated in response to histonedeacetylase inhibition, suppressing ligand sensitivity and regulatingtranscriptional activation by histone deacetylase inhibitors.Conservation of the acetylated ERalpha motif in other nuclear receptorssuggests that acetylation may play an important regulatory role indiverse nuclear receptor signaling functions. A number of structurallydiverse histone deacetylase inhibitors have shown potent antitumorefficacy with little toxicity in vivo in animal models. Severalcompounds are currently in early phase clinical development as potentialtreatments for solid and hematological cancers both as monotherapy andin combination with cytotoxics and differentiation agents.

The HDAC enzyme family constitutes a family of 18 genes that can begrouped into four subclasses; classes I-IV, based on their homology torespective yeast orthologs. HDACs, belonging to classes I, II and IV,comprise 11 members, namely HDAC isoforms 1-11, commonly referred to asthe classical HDACs, are metal-dependent hydrolases. HDACs of class III,which comprise 7 members, known as sirtuins, namely Sirt 1-7, areNAD+-dependent hydrolases. Class I HDACs are nuclear proteins withubiquitous tissue expression. Class II and IV HDACs are found in boththe nucleus and cytoplasm and exhibit tissue-specific expression. TheClass II HDAC family is further subdivided into subclasses IIA and IIB.Class IIA comprises isoforms HDAC4, HDAC5, HDAC7 and HDAC9 while ClassIIB comprises isoforms HDAC6 and HDAC10. HDAC6 contains two tandemdeacetylase domains and a C-terminal zinc finger domain. HDAC10 isstructurally related to HDAC6 but has one additional catalytic domain.Table 1 represents the cellular location and tissue expression ofclassical HDACs (adapted from Witt, O. et al., Cancer Lett., 277:8-21(2008)).

TABLE 1 Classical HDACs, Cellular Location and Tissue Expression ClassIsoform Cellular Location Tissue Expression Class I HDAC 1 NuclearUbiquitous HDAC2 Nuclear Ubiquitous HDAC3 Nuclear Ubiquitous HDAC8Nuclear/ Ubiquitous cytoplasmic Class II A HDAC4 Nuclear/ Heart, smoothcytoplasmic muscles, brain HDAC5 Nuclear/ Heart, smooth cytoplasmicmuscle, brain HDAC7 Nuclear/ Heart, placenta, pancreas, cytoplasmicsmooth muscle HDAC9 Nuclear/ Smooth muscle, brain cytoplasmic Class II BHDAC6 Cytoplasmic Kidney, liver, heart, pancreas HDAC10 CytoplasmicSpleen, kidney, liver Class IV HDAC11 Nuclear/ Heart, smooth muscle,cytoplasmic kidney, brain

HDACs play a significant role in both normal and aberrant cellproliferation and differentiation. HDACs have been associated with somedisease states involving proliferation, including, but not limited to,cell proliferative diseases and conditions, such as various forms ofcancer. (Reviewed in Witt, O. et al., Cancer Lett., 277:8-21 (2008); andPortella A. et al., Nat. Biotechnol., 28:1057-1068 (2010)). Class I andII HDACs have been identified as attractive targets for anticancertherapy. In particular, distinct class I and class II HDAC proteins areoverexpressed in some cancers, including ovarian (HDAC1-3), gastric(HDAC2), and lung cancers (HDAC1 and 3), among others. Also, a possiblecorrelation between HDAC8 and acute myeloid leukemia (AML) has beensuggested. Concerning class II HDAC proteins, aberrant expression ofHDAC6 is induced in some breast cancer cells. Based on their clinicaleffects, HDAC inhibitors have been identified that suppress tumor cellproliferation, induce cell differentiation, and upregulate crucial genesassociated with anti-cancer effects. HDACs have also been implicated invarious types of cancers (Bali P, et al., “Inhibition of histonedeacetylase 6 acetylates and disrupts the chaperone function of heatshock protein 90: A novel basis for antileukemia activity of histonedeacetylase inhibitors,” J. Biol. Chem., 2005 280:26729-26734; Santo L.et al., “Preclinical activity, pharmacodynamic and pharmacokineticproperties of a selective HDAC6 inhibitor, ACY-1215, in combination withbortezomib in multiple myeloma,” Blood, 2012, 119(11): 2579-89),autoimmune or inflammatory diseases (Shuttleworth, S. J., et al., Curr.Drug Targets, 11:1430-1438 (2010)), cognitive and neurodegenerativediseases (Fischer, A., et al., Trends Pharmacol. Sci., 31:605-617(2010); Chuang, D.-M., et al., Trends Neurosci. 32:591-601 (2009)),fibrotic diseases (Pang, M. et al., J. Pharmacol. Exp. Ther.,335:266-272 (2010)), protozoal diseases (see, e.g., U.S. Pat. No.5,922,837), and viral diseases (Margolis, D. M. et al., Curr. Opin. HIVAIDS, 6:25-29 (2011)).

In recent years, there has been an effort to develop HDAC inhibitors ascancer treatments and/or as an adjunct therapy. Mark P A. et al. ExpertOpinion on Investigational Drugs 14 (12): 1497-1511 (2005). The exactmechanisms by which the compounds may work are unclear, but epigeneticpathways have been studied to help elucidate the exact biologicalpathways. Claude Monneret, Anticancer Drugs 18(4):363-370 2007. Forexample, HDAC inhibitors have been shown to induce p21 (WAFT)expression, a regulator of p53's tumor suppressor activity. Rochon V M.et al., Proc. Natl. Acad. Sci. U.S.A. 97(18): 10014-10019, 2000. HDACsare involved in the pathway by which the retinoblastoma protein (pRb)suppresses cell proliferation. The pRb protein is part of a complex thatattracts HDACs to the chromatin so that it will deacetylate histones.Brehm A. et al., Nature 391 (6667): 597-601, 1998. HDAC1 negativelyregulates the cardiovascular transcription factor Kruppel-like factor 5through direct interaction. Matsumura T. et al., J. Biol. Chem. 280(13): 12123-12129, 2005. Estrogen is well-established as a mitogenicfactor implicated in the tumorigenesis and progression of breast cancervia its binding to the estrogen receptor alpha (ERα). Recent dataindicate that chromatin inactivation mediated by HDAC and DNAmethylation is a critical component of ERα silencing its human breastcancer cells. Zhang Z. et al., Breast Cancer Res. Treat. 94(1): 11-16,2005.

Methods of Inhibiting a HDAC Isoform in a Cell

The method comprising contacting the cell with an effective amount ofany one of the disclosed compounds of formula (I), or a pharmaceuticallyacceptable salt form thereof.

In some embodiments, the cell is selected from the group consisting of:a cancer cell, a neuronal cell, a cell of the immune system, a cell ofthe circulatory system, and combinations thereof. In other embodiments,the cell is a cancer cell. In yet other embodiments, the cell is aneuronal cell. In further embodiments, the cell is a cell of the immunesystem. In yet further combinations, the cell is a cell of thecirculatory system.

Non-limiting examples of the cancer cell include: an acute lymphocyticleukemia (ALL) cell, an acute myeloid leukemia (AML) cell, an acutepromyelocytic leukemia (APL) cell, a breast cancer cell, a chronicmyeloid leukemia (CML) cell, a colon cancer cell, a diffuse large B-celllymphoma (DLBCL) cell, a gastrointestinal stromal tumor (GIST) cell, aglioblastoma (GBM) cell, a hepatocellular carcinoma cell, a Hodgkinlymphoma cell, a leukemia cell, a lung cancer cell, a multiple myelomacell, a non-Hodgkin's lymphoma cell, a non-small cell lung cancer(NSCLC) cell, a neuroblastoma cell, an ovarian cancer cell, a pancreaticductal adenocarcinoma cell, a peripheral T-cell lymphoma cell, aprostate cancer cell, a uterine cancer cell, and a Waldenstrom myelomacell, etc.

In some embodiments, the HDAC isoform is selected from the groupconsisting of: HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8,HDAC9, HDAC10, HDAC11, and combinations thereof. In other embodiments,the HDAC isoform is selected from the group consisting of: HDAC1, HDAC3,HDAC6, HDAC10, and combinations thereof.

In some aspects, the compound of formula (I) inhibits the histonedeacetylating activity of the HDAC isoform with a half maximalinhibitory concentration (IC₅₀) of 0.001-4 μM, or any number range inbetween, e.g., 0.001-3.5 μM, 0.002-3.5 μM, 0.002-3 μM, 0.003-3 μM,0.003-2.5 μM, 0.005-2.5 μM, 0.005-2 μM or 0.01-2 μM etc. In otheraspects, the compound of formula (I) inhibits the histone deacetylatingactivity of the HDAC isoform with a half maximal inhibitoryconcentration (IC₅₀) of 0.001-10 μM, or any number range in between,e.g., 0.001-8 μM, 0.002-8 μM, 0.002-6 μM, 0.003-6 μM, 0.003-4 μM,0.005-4 μM, 0.005-2 μM or 0.01-2 μM etc. In yet other aspects, thecompound of formula (I) inhibits the histone deacetylating activity ofthe HDAC isoform with a half maximal inhibitory concentration (IC₅₀) of0.02-10 μM, or any number range in between, e.g., 0.05-10 μM, 0.05-9 μM,0.1-9 μM, 0.1-8 μM, 0.2-8 μM, 0.2-7 μM, 0.4-7 μM or 0.4-6 μM etc. Infurther aspects, the compound of formula (I) inhibits the activity ofthe HDAC isoform with a half maximal inhibitory concentration (IC₅₀) oflower than 10 μM, lower than 8 μM, lower than 6 μM, lower than 4 μM,lower than 2 μM, lower than 1 μM, lower than 0.5 μM, lower than 0.2 μM,lower than 0.1 μM, etc.

In some embodiments, the compound inhibits the histone deacetylatingactivity of the HDAC isoform by at least 90%, at least 80%, at least70%, at least 60%, at least 50%, at least 40%, at least 30%, at least20%, or at least 10%. In other embodiments, the compound inhibits thehistone deacetylating activity of the HDAC isoform by 10-100%, or anypercent range in between, e.g., 10-90%, 15-90%, 30%-90%, 15-80%, 20-80%,30%-80%, 20-70%, 25-70%, 30%-70%, 25-60%, 30-60%, or 30-50%, etc.

In some embodiments, the compound of formula (I) inhibits the histonedeacetylating activity of the HDAC isoform, thereby inhibits cellproliferation, induces cell death, or both.

In some aspects, the method is performed in vitro. A non-limitingexample is a screening assay using the compound of formula (I) as apositive control, a standard, or both to measure the activity of anunknown compound in inhibiting HDAC.

In some aspects, the method is performed in vivo, thereby inhibiting theHDAC isoform in a subject. The contacting is achieved by administeringthe compound, or a pharmaceutically acceptable salt form thereof, in anamount effective to inhibit the HDAC isoform. In other aspects, thesubject is a human, e.g., a patient.

A cancer cell includes a cell derived from a tumor, neoplasm, cancer,precancer, cell line, or any other sources that is potentially capableof unlimited expansion and growth. In some aspects, the cancer cell isderived from a naturally occurring source. In other aspects, the cancercell is artificially created. In some embodiments, the cancer cell iscapable of invasion into a tissue and metastasis when placed into ananimal host. Cancer cells further encompass any malignant cells thathave invaded other tissues, metastasized, or both. In some aspects, oneor more cancer cells of an organism is referred to as a cancer, a tumor,a neoplasm, a growth, a malignancy, or another term used in the artdescribing cells in a cancerous state.

Expansion of a cancer cell includes any process that results in anincrease in the number of individual cells derived from a cancer cell.Expansion of a cancer cell may result from mitotic division,proliferation, or any other form of expansion of a cancer cell, whetherin vitro or in vivo. Expansion of a cancer cell further encompassesinvasion and metastasis. A cancer cell may be in physical proximity tocancer cells from the same clone or from different clones that may ormay not be genetically identical to it. Such aggregations may take theform of a colony, tumor or metastasis, any of which may occur in vivo orin vitro. Slowing the expansion of the cancer cell may be brought abouteither by inhibiting cellular processes that promote expansion or bybringing about cellular processes that inhibit expansion. Processes thatinhibit expansion include processes that slow mitotic division andprocesses that promote cell senescence or cell death. Examples ofspecific processes that inhibit expansion include: caspase dependent andindependent pathways, autophagy, necrosis, apoptosis, and mitochondrialdependent and independent processes and further include any suchprocesses yet to be disclosed.

Addition of a pharmaceutical composition to cancer cells includes allactions by which an effect of the pharmaceutical composition on thecancer cell is realized. The type of addition chosen will depend uponwhether the cancer cells are in vivo, ex vivo, or in vitro, the physicalor chemical properties of the pharmaceutical composition, and the effectthe composition is to have on the cancer cell. Nonlimiting examples ofaddition include addition of a solution including the pharmaceuticalcomposition to tissue culture media in which in vitro cancer cells aregrowing; any method by which a pharmaceutical composition may beadministered to an animal including intravenous, per os, parenteral, orany other of the methods of administration; or the activation orinhibition of cells that in turn have effects on the cancer cells suchas immune cells (e.g., macrophages and CD8⁺ T cells) or endothelialcells that may differentiate into blood vessel structures in the processof angiogenesis or vasculogenesis.

Determination of an effective amount of the disclosed compound is withinthe capability of those skilled in the art, especially in light of thedetailed disclosure provided herein. The effective amount of apharmaceutical composition used to affect a particular purpose as wellas its toxicity, excretion, and overall tolerance may be determined incell cultures or experimental animals by pharmaceutical andtoxicological procedures either known now by those skilled in the art orby any similar method yet to be disclosed. One example is thedetermination of the IC₅₀ (half maximal inhibitory concentration) of thepharmaceutical composition in vitro in cell lines or target molecules.Another example is the determination of the LD₅₀ (lethal dose causingdeath in 50% of the tested animals) of the pharmaceutical composition inexperimental animals. The exact techniques used in determining aneffective amount will depend on factors such as the type andphysical/chemical properties of the pharmaceutical composition, theproperty being tested, and whether the test is to be performed in vitroor in vivo. The determination of an effective amount of a pharmaceuticalcomposition will be well known to one of skill in the art who will usedata obtained from any tests in making that determination. Determinationof an effective amount of disclosed compound for addition to a cancercell also includes the determination of an effective therapeutic amount,including the formulation of an effective dose range for use in vivo,including in humans.

Methods of Treating a HDAC-Associated Disease

Herein the inventors also disclose a method of treating a histonedeacetylase (HDAC)-associated disease, for example, a disease associatedwith a histone deacetylase (HDAC) isoform. Typically the methodcomprises administering to a subject any one of the disclosed compoundsof formula (I), or a pharmaceutically acceptable salt form thereof.

Non-limiting examples of the disease include: a cell proliferativedisease (e.g., cancer), an autoimmune disorder, an inflammatorydisorder, a neurodegenerative disease, and combinations thereof, etc.

In some embodiments, the cell proliferative disease is cancer.Non-limiting examples of cancer include: acute lymphocytic leukemia(ALL), acute myeloid leukemia (AML), acute promyelocytic leukemia (APL),breast cancer, chronic myeloid leukemia (CML), colon cancer, diffuselarge B-cell lymphoma (DLBCL), gastrointestinal stromal tumor (GIST),glioblastoma (GBM), hepatocellular carcinoma, Hodgkin's lymphoma,leukemia, lung cancer, multiple myeloma, non-Hodgkin's lymphoma,non-small cell lung cancer (NSCLC), neuroblastoma, ovarian cancer,pancreatic ductal adenocarcinoma, peripheral T-cell lymphoma, prostatecancer, uterine cancer, Waldenstrom myeloma, and combinations thereof,etc.

In some embodiments, the cancer is selected from the group consistingof: ovarian cancer, prostate cancer, lung cancer, acute myeloidleukemia, multiple myeloma, bladder carcinoma, renal carcinoma, breastcarcinoma, colorectal carcinoma, neuroblastoma, melanoma, gastriccancer, and combinations thereof. In some aspects, the compound offormula (I) inhibits cancer cell proliferation, induces cancer celldeath, or both.

Non-limiting examples of the autoimmune disorder or inflammatorydisorder include: airway hyperresponsiveness, Crohn's disease,inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoidarthritis, systemic lupus erythematosus, type 1 diabetes, ulcerativecolitis, and combinations thereof, etc.

Non-limiting examples of the neurodegenerative disorder include:Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), cerebralischemia, Huntington's disease (HD), Parkinson's disease (PD), spinalmuscular atrophy, and combinations thereof, etc.

In some aspects, the disease is associated with HDAC1, HDAC2, HDAC3,HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, orcombinations thereof. In other aspects, the HDAC-associated disease isassociated with HDAC1, HDAC3, HDAC6, HDAC10, or combinations thereof.

In certain more specific aspects of the invention, the compound offormula (I) inhibits the histone deacetylating activity of the HDACisoform with a half maximal inhibitory concentration (IC₅₀) of 0.001-4μM, or any number range in between, e.g., 0.001-3.5 μM, 0.002-3.5 μM,0.002-3 μM, 0.003-3 μM, 0.003-2.5 μM, 0.005-2.5 μM, 0.005-2 μM or 0.01-2μM etc.

In other aspects, the compound of formula (I) inhibits the histonedeacetylating activity of the HDAC isoform with a half maximalinhibitory concentration (IC₅₀) of 0.001-10 μM, or any number range inbetween, e.g., 0.001-8 μM, 0.002-8 μM, 0.002-6 μM, 0.003-6 μM, 0.003-4μM, 0.005-4 μM, 0.005-2 μM or 0.01-2 μM etc. In yet other aspects, thecompound of formula (I) inhibits the histone deacetylating activity ofthe HDAC isoform with a half maximal inhibitory concentration (IC₅₀) of0.02-10 μM, or any number range in between, e.g., 0.05-10 μM, 0.05-9 μM,0.1-9 μM, 0.1-8 μM, 0.2-8 μM, 0.2-7 μM, 0.4-7 μM or 0.4-6 μM etc. Infurther aspects, the compound of formula (I) inhibits the activity ofthe HDAC isoform with a half maximal inhibitory concentration (IC₅₀) oflower than 10 μM, lower than 8 μM, lower than 6 μM, lower than 4 μM,lower than 2 μM, lower than 1 μM, lower than 0.5 μM, lower than 0.2 μM,lower than 0.1 μM, etc.

In some embodiments, the compound of formula (I) inhibits the histonedeacetylating activity of the HDAC isoform by at least 90%, at least80%, at least 70%, at least 60%, at least 50%, at least 40%, at least30%, at least 20%, or at least 10%. In other embodiments, the compoundinhibits the histone deacetylating activity of the HDAC isoform by10-100%, or any percent range in between, e.g., 10-90%, 15-90%, 30%-90%,15-80%, 20-80%, 30%-80%, 20-70%, 25-70%, 30%-70%, 25-60%, 30-60%, or30-50%, etc.

In yet other aspects, the method further comprises administering achemotherapy drug to the subject. In some embodiments, the chemotherapydrug comprises pomalidomide. In other embodiments, the chemotherapy drugcomprises dexamethasone. In yet other embodiments, the chemotherapy drugcomprises both pomalidomide and dexamethasone.

In some aspects, the composition is administered at 10-400 mg/kg, or anynumber in between, e.g., 10-350 mg/kg, 20-350 mg/kg, 20-300 mg/kg,30-300 mg/kg, 30-250 mg/kg, 40-250 mg/kg, 40-200 mg/kg, 50-200 mg/kg,50-150 mg/kg, 60-150 mg/kg, or 60-100 mg/kg, etc.

In other aspects, the composition is administered about every 4, 8, 12,16, or 24 hours. In yet other aspects, the composition is administeredevery 1-24 hours, or any number in between, e.g., 2-24 hours, 2-18hours, 3-18 hours, 3-16 hours, 4-16 hours, 4-12 hours, 5-12 hours, 5-8hours, etc.

Methods of administration include, but are not limited to, oraladministration and parenteral administration. Parenteral administrationincludes, but is not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural,sublingual, intramucosal, intracerebral, intraventricular, intrathecal,intravaginal, transdermal, rectal, by inhalation, or topically to theears, nose, eyes, or skin. Other methods of administration include butare not limited to infusion techniques including infusion or bolusinjection, by absorption through epithelial or mucocutaneous liningssuch as oral mucosa, rectal and intestinal mucosa. Compositions forparenteral administration may be enclosed in ampoule, a disposablesyringe or a multiple-dose vial made of glass, plastic or othermaterial.

Administration may be systemic or local. Local administration isadministration of the disclosed compound to the area in need oftreatment. Examples include local infusion during surgery, topicalapplication, local injection, or administration by a catheter, by asuppository, or by an implant. Administration may be by direct injectionat the site (or former site) of a cancer, tumor, or precancerous tissueor into the central nervous system by any suitable route, includingintraventricular and intrathecal injection. Intraventricular injectioncan be facilitated by an intraventricular catheter, for example,attached to a reservoir, such as an Ommaya reservoir. Pulmonaryadministration may be achieved by any of a number of methods known inthe art. Examples include use of an inhaler or nebulizer, formulationwith an aerosolizing agent, or via perfusion in a fluorocarbon orsynthetic pulmonary surfactant. The disclosed compound may be deliveredin the context of a vesicle such as a liposome or any other natural orsynthetic vesicle.

A pharmaceutical composition formulated so as to be administered byinjection may be prepared by dissolving the disclosed compound withwater so as to form a solution. In addition, a surfactant may be addedto facilitate the formation of a homogeneous solution or suspension.Surfactants include any complex capable of non-covalent interaction withthe disclosed compound so as to facilitate dissolution or homogeneoussuspension of the compound.

Treatment is contemplated in living entities including but not limitedto mammals (particularly humans) as well as other mammals of economic orsocial importance, including those of an endangered status. Furtherexamples include livestock or other animals generally bred for humanconsumption and domesticated companion animals.

The toxicity and therapeutic efficacy of a pharmaceutical compositionmay be determined by standard pharmaceutical procedures in cell culturesor animals. Examples include the determination of the IC₅₀ (the halfmaximal inhibitory concentration) and the LD₅₀ (lethal dose causingdeath in 50% of the tested animals) for a subject compound. The dataobtained from these cell culture assays and animal studies can be usedin formulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized.

The effective amount of one of the disclosed compounds to result in theslowing of expansion of the cancer cells would preferably result in aconcentration at or near the target tissue that is effective in slowingcellular expansion in neoplastic cells but have minimal effects onnon-neoplastic cells, including non-neoplastic cells exposed toradiation or recognized chemotherapeutic chemical agents. Concentrationsthat produce these effects can be determined using, for example,apoptosis markers such as the apoptotic index and/or caspase activitieseither in vitro or in vivo.

Treatment of a condition is the practice of any method, process, orprocedure with the intent of halting, inhibiting, slowing or reversingthe progression of a disease, disorder or condition, substantiallyameliorating clinical symptoms of a disease disorder or condition, orsubstantially preventing the appearance of clinical symptoms of adisease, disorder or condition, up to and including returning thediseased entity to its condition prior to the development of thedisease.

The addition of a therapeutically effective amount of one of thedisclosed compounds encompasses any method of dosing of a compound.Dosing of the disclosed compound may include single or multipleadministrations of any of a number of pharmaceutical compositions thatinclude the disclosed compound as an active ingredient. Examples includea single administration of a slow release composition, a course oftreatment involving several treatments on a regular or irregular basis,multiple administrations for a period of time until a diminution of thedisease state is achieved, preventative treatments applied prior to theinstigation of symptoms, or any other dosing regimen known in the art oryet to be disclosed that one skilled in the art would recognize as apotentially effective regimen. A final dosing regimen including theregularity of and mode of administration will be dependent on any of anumber of factors including but not limited to the subject beingtreated; the severity of the affliction; the manner of administration,the stage of disease development, the presence of one or more otherconditions such as pregnancy, infancy, or the presence of one or moreadditional diseases; or any other factor now known or yet to bedisclosed that affects the choice of the mode of administration, thedose to be administered and the time period over which the dose isadministered.

Pharmaceutical compositions that include one of the disclosed compoundsmay be administered prior to, concurrently with, or after administrationof a second pharmaceutical composition that may or may not include thecompound. If the compositions are administered concurrently, they areadministered within one minute of each other. If not administeredconcurrently, the second pharmaceutical composition may be administereda period of one or more minutes, hours, days, weeks, or months before orafter the pharmaceutical composition that includes the compound.Alternatively, a combination of pharmaceutical compositions may becyclically administered. Cycling therapy involves the administration ofone or more pharmaceutical compositions for a period of time, followedby the administration of one or more different pharmaceuticalcompositions for a period of time and repeating this sequentialadministration, in order to reduce the development of resistance to oneor more of the compositions, to avoid or reduce the side effects of oneor more of the compositions, and/or to improve the efficacy of thetreatment.

The invention further encompasses kits that facilitate theadministration of one of the disclosed compounds to a diseased entity.An example of such a kit includes one or more unit dosages of thecompound. The unit dosage would be enclosed in a preferably sterilecontainer and would be comprised of the disclosed compound and apharmaceutically acceptable carrier. In another aspect, the unit dosagewould comprise one or more lyophilates of the compound. In this aspectof the invention, the kit may include another preferably sterilecontainer enclosing a solution capable of dissolving the lyophilate.However, such a solution need not be included in the kit and may beobtained separately from the lyophilate. In another aspect, the kit mayinclude one or more devices used in administrating the unit dosages or apharmaceutical composition to be used in combination with the compound.Examples of such devices include, but are not limited to, a syringe, adrip bag, a patch or an enema. In some aspects of the invention, thedevice comprises the container that encloses the unit dosage.

Pharmaceutical compositions including one of the disclosed compounds maybe used in methods of treating cancer. Such methods involve theadministration of a therapeutic amount of a pharmaceutical compositionthat includes the disclosed compound and/or a pharmaceuticallyacceptable salt thereof to a mammal, preferably a mammal in which acancer has been diagnosed.

A therapeutic amount further includes the prevention of progression ofthe cancer to a neoplastic, malignant or metastatic state. Suchpreventative use is indicated in conditions known or suspected ofpreceding progression to neoplasia or cancer, in particular, wherenon-neoplastic cell growth consisting of hyperplasia, metaplasia, ormost particularly, dysplasia has occurred (for review of such abnormalgrowth conditions, see Robbins and Angell, 1976, Basic Pathology, 2dEd., W. B. Saunders Co., Philadelphia, pp. 68-79). Hyperplasia is a formof controlled cell proliferation involving an increase in cell number ina tissue or organ, without significant alteration in structure oractivity. For example, endometrial hyperplasia often precedesendometrial cancer and precancerous colon polyps often transform intocancerous lesions. Metaplasia is a form of controlled cell growth inwhich one type of adult or fully differentiated cell substitutes foranother type of adult cell. Metaplasia can occur in epithelial orconnective tissue cells. A typical metaplasia involves a somewhatdisorderly metaplastic epithelium. Dysplasia is frequently a forerunnerof cancer, and is found mainly in the epithelia; it is the mostdisorderly form of non-neoplastic cell growth, involving a loss inindividual cell uniformity and in the architectural orientation ofcells. Dysplastic cells often have abnormally large, deeply stainednuclei, and exhibit pleomorphism. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation, and is oftenfound in the cervix, respiratory passages, oral cavity, and gallbladder.

Alternatively, or in addition to the presence of abnormal cell growthcharacterized as hyperplasia, metaplasia, or dysplasia, the presence ofone or more characteristics of a transformed phenotype or of a malignantphenotype, displayed in vivo or displayed in vitro by a cell samplederived from a patient can indicate the desirability ofprophylactic/therapeutic administration of the pharmaceuticalcomposition that includes the compound. Such characteristics of atransformed phenotype include morphology changes, looser substratumattachment, loss of contact inhibition, loss of anchorage dependence,protease release, increased sugar transport, decreased serumrequirement, expression of fetal antigens, disappearance of the 250,000dalton cell surface protein, etc. (see also id., at pp. 84-90 forcharacteristics associated with a transformed or malignant phenotype).Further examples include leukoplakia (a benign-appearing hyperplastic ordysplastic lesion of the epithelium) or Bowen's disease (a carcinoma insitu), which are pre-neoplastic lesions indicating the desirability ofprophylactic intervention. In another example, fibrocystic disease,including cystic hyperplasia, mammary dysplasia, adenosis, or benignepithelial hyperplasia, indicates the desirability of prophylacticintervention.

In some aspects of the invention, use of the disclosed compound may bedetermined by one or more physical factors such as tumor size and gradeor one or more molecular markers and/or expression signatures thatindicate prognosis and the likely response to treatment with thecompound. For example, determination of estrogen (ER) and progesterone(PR) steroid hormone receptor status has become a routine procedure inassessment of breast cancer patients. See, for example, Fitzgibbons etal, Arch. Pathol. Lab. Med. 124:966-78, 2000. Tumors that are hormonereceptor positive are more likely to respond to hormone therapy and alsotypically grow less aggressively, thereby resulting in a betterprognosis for patients with ER⁺/PR⁺ tumors. In a further example,overexpression of human epidermal growth factor receptor 2 (HER-2/neu),a transmembrane tyrosine kinase receptor protein, has been correlatedwith poor breast cancer prognosis (see, e.g., Ross et al, The Oncologist8:307-25, 2003), and Her-2 expression levels in breast tumors are usedto predict response to the anti-Her-2 monoclonal antibody therapeutictrastuzumab (Herceptin®, Genentech, South San Francisco, Calif.).

In another aspect of the invention, the diseased entity exhibits one ormore predisposing factors for malignancy that may be treated byadministration of a pharmaceutical composition including the compound.Such predisposing factors include but are not limited to chromosomaltranslocations associated with a malignancy such as the Philadelphiachromosome for chronic myelogenous leukemia and t(14;18) for follicularlymphoma; an incidence of polyposis or Gardner's syndrome that areindicative of colon cancer; benign monoclonal gammopathy which isindicative of multiple myeloma; kinship with persons who have had orcurrently have a cancer or precancerous disease; exposure tocarcinogens; or any other predisposing factor that indicates inincreased incidence of cancer now known or yet to be disclosed.

The invention further encompasses methods of treating cancer thatcomprise combination therapies, wherein the combination therapiescomprise the administration of a pharmaceutical composition includingone of the disclosed compounds and another treatment modality. Suchtreatment modalities include, but are not limited to, radiotherapy,chemotherapy, surgery, immunotherapy, cancer vaccines,radioimmunotherapy, treatment with pharmaceutical compositions otherthan those which include the disclosed compound, or any other methodthat effectively treats cancer in combination with the disclosedcompound now known or yet to be disclosed. Combination therapies may actsynergistically. That is, the combination of the two therapies is moreeffective than either therapy administered alone. This results in asituation in which lower dosages of both treatment modalities may beused effectively. This in turn reduces the toxicity and side effects, ifany, associated with the administration either modality without areduction in efficacy.

In another aspect of the invention, the pharmaceutical compositionincluding one of the disclosed compounds is administered in combinationwith a therapeutically effective amount of radiotherapy. Radiotherapymay be administered concurrently with, prior to, or following theadministration of the pharmaceutical composition including the compound.Radiotherapy may act additively or synergistically with thepharmaceutical composition including the compound. This particularaspect of the invention would be most effective in cancers known to beresponsive to radiotherapy. Cancers known to be responsive toradiotherapy include, but are not limited to, Non-Hodgkin's lymphoma,Hodgkin's disease, Ewing's sarcoma, testicular cancer, prostate cancer,ovarian cancer, bladder cancer, larynx cancer, cervical cancer,nasopharynx cancer, breast cancer, colon cancer, pancreatic cancer, headand neck cancer, esophageal cancer, rectal cancer, small-cell lungcancer, non-small cell lung cancer, brain tumors, other CNS neoplasms,or any other such tumor now known or yet to be disclosed.

Examples of pharmaceutical compositions that may be used in combinationwith one of the disclosed compounds include nucleic acid bindingcompositions, such as cis-diamminedichloro platinum (II) (cisplatin),doxorubicin, 5-fluorouracil, taxol, and topoisomerase inhibitors such asetoposide, teniposide, irinotecan, and topotecan. Still otherpharmaceutical compositions include antiemetic compositions, such asmetoclopromide, domperidone, prochlorperazine, promethazine,chlorpromazine, trimethobenzamide, ondansetron, granisetron,hydroxyzine, acethylleucine monoethanolamine, alizapride, azasetron,benzquinamide, bietanautine, bromopride, buclizine, clebopride,cyclizine, dimenhydrinate, diphenidol, dolasetron, meclizine,methallatal, metopimazine, nabilone, oxyperndyl, pipamazine,scopolamine, sulpiride, tetrahydrocannabinols, thiethylperazine,thioproperazine, and tropisetron.

Still other examples of pharmaceutical compositions that may be used incombination with the pharmaceutical composition including one of thedisclosed compounds are hematopoietic colony stimulating factors.Examples of hematopoietic colony stimulating factors include, but arenot limited to, filgrastim, sargramostim, molgramostim, and epoietinalfa. Alternatively, the pharmaceutical composition including one of thedisclosed compounds may be used in combination with an anxiolytic agent.Examples of anxiolytic agents include, but are not limited to,buspirone, and benzodiazepines such as diazepam, lorazepam, oxazapam,chlorazepate, clonazepam, chlordiazepoxide and alprazolam.

Pharmaceutical compositions that may be used in combination withpharmaceutical compositions that include one of the disclosed compoundsmay include analgesic agents. Such agents may be opioid or non-opioidanalgesic. Non-limiting examples of opioid analgesics include morphine,heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, metopon,apomorphine, normorphine, etorphine, buprenorphine, meperidine,lopermide, anileridine, ethoheptazine, piminidine, betaprodine,diphenoxylate, fentanil, sufentanil, alfentanil, remifentanil,levorphanol, dextromethorphan, phenazocine, pentazocine, cyclazocine,methadone, isomethadone, and propoxyphene. Suitable non-opioid analgesicagents include, but are not limited to, aspirin, celecoxib, rofecoxib,diclofinac, diflusinal, etodolac, fenoprofen, flurbiprofen, ibuprofen,ketoprofen, indomethacin, ketorolac, meclofenamate, mefanamic acid,nabumetone, naproxen, piroxicam, sulindac, or any other analgesic nowknown or yet to be disclosed.

In other aspects of the invention, pharmaceutical compositions includingone of the disclosed compounds may be used in combination with a methodthat involves treatment of cancer ex vivo. One example of such atreatment is an autologous stem cell transplant. In this method, adiseased entity's autologous hematopoietic stem cells are harvested andpurged of all cancer cells. A therapeutic amount of a pharmaceuticalcomposition including one of the disclosed compounds may then beadministered to the patient prior to restoring the entity's bone marrowby addition of either the patient's own or donor stem cells.

Cancers that may be treated by pharmaceutical compositions including theone of the disclosed compounds either alone or in combination withanother treatment modality include solid tumors such as fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer,kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovariancancer, prostate cancer, esophageal cancer, stomach cancer, oral cancer,nasal cancer, throat cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterinecancer, testicular cancer, small cell lung carcinoma, bladder carcinoma,lung cancer, epithelial carcinoma, glioma, glioblastoma multiforme,astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skincancer, melanoma, neuroblastoma, and retinoblastoma.

Additional cancers that may be treated by pharmaceutical compositionsincluding the disclosed compound include blood borne cancers such asacute lymphoblastic leukemia (“ALL”), acute lymphoblastic B-cellleukemia, acute lymphoblastic T-cell leukemia, acute myeloblasticleukemia (“AML”), acute promyelocytic leukemia (“APL”), acutemonoblastic leukemia, acute erythroleukemic leukemia, acutemegakaryoblastic leukemia, acute myelomonocytic leukemia, acutenonlymphocyctic leukemia, acute undifferentiated leukemia, chronicmyelocytic leukemia (“CML”), chronic lymphocytic leukemia (“CLL”), hairycell leukemia, multiple myeloma, lymphoblastic leukemia, myelogenousleukemia, lymphocytic leukemia, myelocytic leukemia, Hodgkin's disease,non-Hodgkin's Lymphoma, Waldenstrom's macroglobulinemia, Heavy chaindisease, and Polycythemia vera.

Examples that represent different aspects of the invention follow. Suchexamples should not be construed as limiting the scope of thedisclosure. Alternative mechanistic pathways and analogous structureswithin the scope of the invention would be apparent to those skilled inthe art.

EXAMPLES

Elements and acts in the example are intended to illustrate theinvention for the sake of simplicity and have not necessarily beenrendered according to any particular sequence or embodiment. The exampleis also intended to establish possession of the invention by theInventors.

Example 1. Example Compounds of Formula (I) or Formula (II)4-((1-(cyclohexylmethyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #1)

4-((1-cyclohexyl-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide (ID#2)

4-((1-cycloheptyl-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide (ID#3)

4-((1-(1-fluorocyclohexyl)methyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #4)

4-((1-(cyclopentylmethyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #5)

4-((1-(2-cyclopentylethyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamideID #6

4-((1-(4,4-difluorocyclohexyl)methyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #7)

4-((1-(4,4-difluorocyclohexyl)methyl)-5-fluoro-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #8)

N-hydroxy-4-((1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide(ID #9)

4-((5-fluoro-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #10)

Example 2. Cell Viability Assays with MM1.S Cells

Cell viability in the presence of varying concentrations of the abovelisted compounds at different time points was used to assesscytotoxicity and the effect of the compounds on cell proliferation. IC₅₀(or percent activity) data for the disclosed compounds in the MM1.S cellline are summarized in Table 2.

Cell Viability Assay—Cell viability was measured by the CellTiter-GlO®cell viability assay from Promega (Madison, Wis.). The CellTiter-GlO®Luminescent Cell Viability Assay is a homogeneous method to determinethe number of viable cells in culture based on quantitation of the ATPpresent, which signals the presence of metabolically active cells.Following treatment, CellTiter-GlO® is added to treatment wells andincubated at 37° C. luminescence values were measured at using aMolecular Devices Spectramax microplate reader

Single Agent Studies—Cells were grown to 70% confluency, trypsinized,counted, and seeded in 96 well flat-bottom plates at a finalconcentration of 2.5×10³-5×10³ cells/well (Day 0). Cells were allowed toincubate in growth media for 24 hours. Treatment with the test agents orstandard agents began on Day 1 and continued for 72 hours. At the 72hour timepoint, treatment containing media was removed. Viable cellnumbers are quantified by the CellTiter-GlO® cell viability assay asdescribed above. Results from these studies were used to calculate anIC₅₀ value (concentration of drug that inhibits cell growth by 50percent of control) for each compound.

Data Collection—For single agent and combination studies, data from eachexperiment was collected and expressed as % Cell Growth using thefollowing calculation:

Cell Growth=(f _(test) /f _(vehicle))×100

Where f_(test) is the fluorescence of the tested sample, and f_(vehicle)is the fluorescence of the vehicle in which the drug is dissolved. Doseresponse graphs and IC₅₀ values were generated using Prism 6 software(GraphPad) using the following equation:

$Y = \frac{\left( {{Top}\text{-}{Bottom}} \right)}{\left( {1 + {10^{({{({{\log \; {IC}\; 50} - X})} - {HillSlope}})}}} \right)}$

Where X is the logarithm of concentration and Y is the response. Ystarts at the Bottom and goes to Top with a sigmoid shape.

TABLE 2 IC₅₀ of example compounds in MM1.S cells Compound IC₅₀ in ID #MM1.S (μM)  1 0.5  2 2.0  3 2.1  4 2.6  5 2.2  6 1.1  7 0.7  8 1.2  92.9 10 2.9

Example 3. Cell Viability Assays with Various Cell Lines

Compound ID #1 (a compound of formula (I)) and Compound ID #3 (acompound of formula (II)) were tested for the inhibition of cancer cellproliferation. Cell viability in the presence of varying concentrationsof Compound ID #1 and Compound ID #3 at different time points was usedto assess cytotoxicity and the effect of the compounds on cellproliferation. The 50% inhibitory concentration (IC50) data for thecompounds is summarized in Table 3. The data clearly show the surprisingand unexpected increased anti-cancer activity associated with CompoundID #1 compared to Compound ID #3.

Cell Viability Assay—Cell viability was measured by the CellTiter-Glo®cell viability assay Promega (Madison, Wis.). The CellTiter-Glo®Luminescent Cell Viability Assay is a homogeneous method to determinethe number of viable cells in culture based on quantitation of the ATPpresent, which signals the presence of metabolically active cells.Following treatment, CellTiter-Glo® is added to treatment wells andincubated at 37° C. luminescence values were measured at using aMolecular Devices Spectramax microplate reader.

Single Agent Studies—Cells were grown to 70% confluency, trypsinized,counted, and seeded in 96 well flat-bottom plates at a finalconcentration of 2.5×10³-5×10³ cells/well (Day 0). Cells were allowed toincubate in growth media for 24 hours. Treatment with the test agentsbegan on Day 1 and continued for 72 hours. At the 72-hour time point,treatment-containing media was removed. Viable cell numbers arequantified by the CellTiter-Glo® cell viability assay as describedabove. Experiments were run with triplicate concentrations to determinegrowth inhibitory activity. Results from these studies were used tocalculate an IC₅₀ value (concentration of drug that inhibits cell growthby 50 percent of control) for each compound.

Data Collection—For single agent and combination studies, data from eachexperiment was collected and expressed as % Cell Growth using thefollowing calculation:

% Cell Growth=(f _(test) /f _(vehicle))×100

Where f_(test) is the luminescence of the tested sample, and f_(vehicle)is the luminescence of the vehicle in which the drug is dissolved. Doseresponse graphs and IC₅₀ values were generated using Prism 6 software(GraphPad).

TABLE 3 ID #1 ID #3 Improvement ID #1 Tumor IC₅₀ IC₅₀ over ID #3 TypeCell line (μM) (μM) fold Percent GBM U251 1.58  4.07  2.58 61.18% BreastMDA-157 2.75  7.59  2.76 63.77% Breast MDA468 3.86  9.33  2.42 58.63%Breast MCF7 1.96  3.55  1.81 44.79% GIST GIST48 1.32  4.24  3.21 68.87%GIST GIST882 5.75 17.78  3.09 67.66% Uterine AN3CA 1.60  4.57  2.8664.99% Uterine MFE280 5.01  9.71  1.94 48.40% Uterine SKUT-1 3.80  9.55 2.51 60.21% Uterine SKUT-1B 2.51  6.92  2.76 63.73% Uterine MFE296 4.3712.59  2.88 65.29% Uterine Ishikawa 1.70  4.37  2.57 61.10% UterineSNG-M 1.70  8.07  4.75 78.93% Myeloma H929 0.83  1.38  1.66 39.86%Myeloma MM1.S 0.50  2.09  4.18 76.08% Myeloma KMS-11 1.42  3.02  2.1352.98% Myeloma KMS-34 0.61  1.36  2.23 55.15% Myeloma RPMI- 0.66  1.51 2.29 56.29% 8226 Myeloma U266 1.17  4.1  3.50 71.46% ALL RS4-11 1.10 2.44  2.22 54.92% AML MV411 0.63  2.75  4.37 77.09% CML K562 1.58  5.25 3.32 69.90% Lymphoma SUDHL-4 0.24  4.17 17.38 94.24% Lymphoma SUDHL-100.55  1.51  2.75 63.58% Lymphoma OCI-LY3 1.05  2.29  2.18 54.15%Lymphoma RAMOS 1.38  2.88  2.09 52.08% Lymphoma Raji 1.83  4.83  2.6462.11% Lymphoma Mino 1.20  4.17  3.48 71.22% Lymphoma BC-1 1.79  4.93 2.75 63.69% Lymphoma JEKO 0.97  2.22  2.29 56.31% Lymphoma Toledo 0.81 3.21  3.96 74.77% NSCLC A549 2.29 10  4.37 77.10% NSCLC H1650 6.61 18.2 2.75 63.68% NSCLC H460 3.47 10.47  3.02 66.86% NSCLC H1437 2.19  4.79 2.19 54.28% Ovarian ES2 3.98 13.18  3.31 69.80% Ovarian A2780 2.03 5.58  2.75 63.62%

Example 4. In Vivo Screening in a Model of Human Myeloma Single AgentScreening

In vivo efficacy studies of Compound ID #1, Compound ID #2, and CompoundID #3 were tested in human MM1S xenograft model as shown in FIG. 1.Female athymic nude mice were inoculated with 5.0×10⁶ MM1S human myelomacells suspended in a mixture of 50% Matrigel and 50% tissue culturemedia in a total volume of 100 μL. Eighteen days following inoculation,the mice were pair-matched into four groups of five mice per group at anaverage tumor volume of 171 mm³ per group. Group 1 (G1) was treated withvehicle only daily for 19 days. Group 2 (G2) was treated with CompoundID #1 at 100 mg/kg daily for 19 days. Group 3 (G3) was treated withCompound ID #2 at 100 mg/kg daily for 19 days. Group 4 (G4) was treatedwith Compound ID #3 at 100 mg/kg daily for 19 days. Vehicle and CompoundID #1, Compound ID #2 and Compound ID #3 were administered orally viaoral gavage. Body weights and tumor measurements were collected twiceweekly. Tumor width and length were measured in millimeters andconverted to tumor volume (in cubic millimeters) using the formula:

${{tumor}\mspace{14mu} {volume}\mspace{14mu} \left( {mm}^{3} \right)} = {\frac{{width}^{2} \times {length}}{2}.}$

Compound ID #1 demonstrated significantly superior anticancer activitywhen compared to either Compound ID #2 or Compound ID #3 (FIG. 1).

2. Combination Screening

Compound ID #1, Compound ID #2 and Compound ID #3 were tested incombination with the FDA approved anticancer drug Pomalyst(pomalidomide) (FIG. 2). Female athymic nude mice were inoculated with5.0×10⁶ MM1.S human myeloma cells suspended in a mixture of 50% Matrigeland 50% tissue culture media in a total volume of 100 μL. Eighteen daysfollowing inoculation, the mice were pair-matched into four groups offive mice per group at an average tumor weight of 171 mm³ per group.Group 1 (G1) was treated with vehicle only daily for 19 days. Group 2(G2) was treated orally with pomalidomide at 10 mg/kg andintraperitoneally with dexamethasone at 0.3 mg/kg daily for 4 days perweek for to Day 19. Group 3 (G3) was treated with Compound ID #1 at 100mg/kg daily for 19 days. Group 4 (G4) was treated with Compound ID #2 at100 mg/kg daily for 19 days. Group 5 (G5) was treated with Compound ID#3 at 100 mg/kg daily for 19 days. Group 6 (G6) was treated withCompound ID #1 at 100 mg/kg daily for 19 days pluspomalidomide/dexamethasone. Group 7 (G7) was treated with Compound ID #2at 100 mg/kg daily for 19 days plus pomalidomide/dexamethasone. Group 8(G8) was treated with Compound ID #3 at 100 mg/kg daily for 19 days pluspomalidomide/dexamethasone. Vehicle and Compound ID #1, Compound ID #2and Compound ID #3 were administered orally via oral gavage. Bodyweights and tumor measurements were collected twice weekly. Tumor widthand length were measured in millimeters and converted to tumor volume(in cubic millimeters) using the formula:

${{tumor}\mspace{14mu} {volume}\mspace{14mu} \left( {mm}^{3} \right)} = {\frac{{width}^{2} \times {length}}{2}.}$

Compound ID #1 demonstrated significantly superior anticancer activitywhen combined with pomalidomide/dexamethasone compared to eitherCompound ID #2 or ID #3 when combined with pomalidomide/dexamethasone(FIG. 2).

Example 5. In Vitro HDAC Enzyme Inhibition

Purified histone deacetylase enzymes 1, 3, 6, 10 were incubated withfluorescently labeled substrate and test compounds in a standardizedreaction mixture in 384 well plates. Upon termination of the reaction,samples were introduced onto microfluidic chips. Samples migrate throughchannels in the chips and product and substrate are separated based onthe difference in their charge and mass (electrophoretic mobilityshift). Enzyme activity is measured by direct comparison of thefluorescence in the product and substrate peaks and the resultspresented in Table 4.

TABLE 4 HDAC1 HDAC3 HDAC6 HDAC10 Compound (IC₅₀ μM) (IC₅₀ μM) (IC₅₀ μM)(IC₅₀ μM) ID #1 0.130 0.019 0.0064 0.137 ID #2 0.385 0.068 0.015 3.56 ID#3 0.445 0.079 0.0145 3.12

Compound ID #1 demonstrated more potent inhibition of the HDAC isoformstested.

Example 6. Pharmacokinetic Studies

Plasma samples were collected from healthy mice and analyzed for levelsof compound ID #1, ID #2, and ID #3 and reported in Table 5. In brief,compounds were individually and accurately weighed out to produce astock solution in Dimethyl-sulfoxide (DMSO) at a concentration of 2mg/mL and stored at −20° C. From the stock solution, a working stock wasprepared by diluting it in Methanol-Water 50:50 (v/v) at 20 μg/mL forcalibration curve preparation and stored at 4° C. Standards used forquantitation and quality control samples (QC's) were prepared on thesame day of sample processing using blank plasma obtained fromnon-treated mice. For each analyte, standards were prepared throughserial dilution of the working stock at concentrations of 0.1, 0.5, 1.0,10, 50, 100, 500 and 1000 ng/mL; QC's were prepared at intermediateconcentrations of 0.75, 7.5, 75 and 750 ng/mL. Plasma samples werestored at −80° C. until ready for analysis and then placed on ice forthawing. An aliquot of 20 μL for samples, standards and QC's, weretransferred into a 96-well extraction plate according to a pre-definedlayout. Appropriate volume of Methanol-Formic Acid 99.9-0.1 containinginternal standard (Verapamil at 25 ng/mL) was added to each well andsamples were extracted under vacuum. Eluent was then transferred into aLCMS plate for analysis using a suitable column for separation and MRMdetection. Concentration of analytes was calculated based on thecalibration curve and analyzed for pharmacokinetic parameters by usingNon-compartmental analysis (NCA) using WinNonlin Phoenix software.Parameters such as Cmax, Tmax, half-life, AUC(0-last), AUC(0-∞), volumeof distribution (Vss) and clearance (Cl/F) were reported.

The analogs were tested for pharmacokinetic analysis in the mouse withdoses of 1 mg/kg for intravenous (IV) route and 5 mg/kg for oral (P0)route. The IV dose showed that out of the three analogs, compound ID #1showed a longer half-life than did the other 2 analogs (3.7 hr versus1.2 and 2.37 hr, respectively). Overall exposure following IV dose alsofavored compound ID #1 with AUCO-∞ 1488.1 hr*ng/mL whereas compound ID#2 and ID #3 were 329.1 and 1193.7 hr*ng/mL, respectively.

Similarly, following oral dose all three compounds showed rapidabsorption (<1 hr) with compound ID #1 showing the highest maximalconcentration at 757.4 ng/mL, whereas compounds ID #2 and ID #3 achieved407.1 and 348.6 ng/mL, respectively. Overall exposure following oraldoses also clearly distinguished compound ID #1 with AUCO-∞ at 3974.8ng/mL*hr, while compounds ID #2 and ID #3 showed 1277.3 and 1648.6ng/mL*hr, respectively.

TABLE 5 Dose (mg/kg) - Route ID#1 ID#2 ID#3 1.0 5.0 1.0 5.0 1.0 5.0 PKmg/kg mg/kg mg/kg mg/kg mg/kg mg/kg parameters IV PO IV PO IV PO Rsq(>0.85) 0.97 0.99 0.85 1.00 0.92 1.00 Half-life (hr) 3.70 3.39 1.20 2.562.37 2.99 Tmax (hr) 0.08 0.50 0.08 0.50 0.08 0.25 Cmax 1288.53 757.43508.17 407.10 829.80 348.57 (ng/mL) AUC_(0-last) 1444.47 3222.99 324.401275.41 1191.74 1642.27 (hr*ng/mL) AUC_(0-∞) 1488.05 3974.82 329.141277.26 1193.68 1648.60 (hr*ng/mL) AUC % 0.25 0.75 1.44 0.14 0.16 0.38Extrap Vss 1.21 — 3.50 — 1.96 — (L/kg) Vz/F obs — 6.16 — 14.48 — 13.09(L/kg) Cl/F obs 0.69 1.26 3.04 3.91 0.84 3.03 (L/hr/kg) % F — 53.42 —77.61 — 27.62 Permeabiltiy 16.75 nm/s 16.51 nm/s 22.01 nm/s (PAMPA) %PPB 99.55; 99.83; N/A 99.48; 99.45; (m, r, hu) 99.44 99.36 S9 in vitro74.82 min >90 min 72.91 half-life

Unless defined otherwise, all technical and scientific terms herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. All publications, patents, andpatent publications cited are incorporated by reference herein in theirentirety for all purposes.

It is understood that the disclosed invention is not limited to theparticular methodology, protocols and materials described as these canvary. It is also understood that the terminology used herein is for thepurposes of describing particular embodiments only and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1-38. (canceled)
 39. A method of inhibiting the histone deacetylatingactivity of a histone deacetylase (HDAC) isoform in a cell, comprisingcontacting the cell with a compound of formula (I):

wherein: X is selected from the group consisting of: H, halo, —C₁-C₆alkyl, aryl, —C₃-C₇ cycloalkyl, and -3- to 10-membered heterocycle, anyof which is unsubstituted, or substituted with one or more of: -halo,—C₁-C₆ alkyl, —O—(C₁-C₆ alkyl), —OH, —CN, —COOR′, —OC(O)R′, NHR′,N(R′)₂, —NHC(O)R′, and —C(O)NHR′, wherein R′ is —H or —C₁-C₆ alkyl; A isselected from the group consisting of: a bond, —C₁-C₆ alkyl, and —C₃-C₇cycloalkyl, any of which is unsubstituted, or substituted with one ormore of: -halo, —C₁-C₆ alkyl, —O—(C₁-C₆ alkyl), —OH, —CN, —COOR′,—OC(O)R′, NHR′, N(R′)₂, —NHC(O)R′, and —C(O)NHR′, wherein R′ is —H or—C₁-C₆ alkyl; Y is selected from the group consisting of: H, —C₁-C₆alkyl, —C₃-C₇ cycloalkyl, aryl, and -3- to 10-membered heterocycle, anyof which is unsubstituted, or substituted with one or more of: -halo,—C₁-C₆ alkyl, —O—(C₁-C₆ alkyl), —OH, —CN, —COOR′, —OC(O)R′, NHR′,N(R′)₂, —NHC(O)R′, and —C(O)NHR′, wherein R′ is —H or —C₁-C₆ alkyl; andQ is selected from the group consisting of: —H, -halo, —C₁-C₆ alkyl,—O—(C₁-C₆ alkyl), —OH, —CN, —COOR′, —OC(O)R′, NHR′, N(R′)₂, —NHC(O)R′,and —C(O)NHR′, wherein R′ is —H or —C₁-C₆ alkyl; wherein the compoundinhibits the histone deacetylating activity of the HDAC isoform in thecell.
 40. The method of claim 39, wherein X is H or halo; Q is H; and Ais a bond or —C₁-C₆ alkyl.
 41. The method of claim 40, wherein Y is—C₃-C₇ cycloalkyl, unsubstituted, or substituted with one or more of:-halo and -3- to 10-membered heterocycle.
 42. The method of claim 39,wherein the compound is selected from the group consisting of:4-((1-(cyclohexylmethyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #1)

4-((1-cyclohexyl-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide (ID#2)

4-((1-cycloheptyl-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide (ID#3)

4-((1-((1-fluorocyclohexyl)methyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #4)

4-((1-(cyclopentylmethyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #5)

4-((1-(2-cyclopentylethyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #6)

4-((1-((4,4-difluorocyclohexyl)methyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #7)

4-((1-((4,4-difluorocyclohexyl)methyl)-5-fluoro-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #8)

N-hydroxy-4-((1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide(ID #9)

and4-((5-fluoro-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #10)


43. The method of claim 39, further comprising determining an expressionsignature in the cell that indicates the likely response to treatmentwith the compound, wherein the expression signature comprises expressionof the HDAC isoform.
 44. The method of claim 43, wherein the HDACisoform is selected from the group consisting of: HDAC1, HDAC3, HDAC6,and HDAC10.
 45. The method of claim 39, wherein the cell is selectedfrom the group consisting of: a cancer cell, a neuronal cell, a cell ofthe immune system, a cell of the circulatory system, and combinationsthereof.
 46. A method of treating a subject having a disease byinhibiting an HDAC isoform, comprising administering to the subject acompound of formula (I):

wherein: X is selected from the group consisting of: H, halo, —C₁-C₆alkyl, aryl, —C₃-C₇ cycloalkyl, and -3- to 10-membered heterocycle, anyof which is unsubstituted, or substituted with one or more of: -halo,—C₁-C₆ alkyl, —O—(C₁-C₆ alkyl), —OH, —CN, —COOR′, —OC(O)R′, NHR′,N(R′)₂, —NHC(O)R′, and —C(O)NHR′, wherein R′ is —H or —C₁-C₆ alkyl; A isselected from the group consisting of: a bond, —C₁-C₆ alkyl, and —C₃-C₇cycloalkyl, any of which is unsubstituted, or substituted with one ormore of: -halo, —C₁-C₆ alkyl, —O—(C₁-C₆ alkyl), —OH, —CN, —COOR′,—OC(O)R′, NHR′, N(R′)₂, —NHC(O)R′, and —C(O)NHR′, wherein R′ is —H or—C₁-C₆ alkyl; Y is selected from the group consisting of: H, —C₁-C₆alkyl, —C₃-C₇ cycloalkyl, aryl, and -3- to 10-membered heterocycle, anyof which is unsubstituted, or substituted with one or more of: -halo,—C₁-C₆ alkyl, —O—(C₁-C₆ alkyl), —OH, —CN, —COOR′, —OC(O)R′, NHR′,N(R′)₂, —NHC(O)R′, and —C(O)NHR′, wherein R′ is —H or —C₁-C₆ alkyl; andQ is selected from the group consisting of: —H, -halo, —C₁-C₆ alkyl,—O—(C₁-C₆ alkyl), —OH, —CN, —COOR′, —OC(O)R′, NHR′, N(R′)₂, —NHC(O)R′,and —C(O)NHR′, wherein R′ is —H or —C₁-C₆ alkyl; wherein the compoundinhibits the histone deacetylating activity of the HDAC isoform.
 47. Themethod of claim 46, wherein X is H or halo; Q is H; and A is a bond or—C₁-C₆ alkyl.
 48. The method of claim 47, wherein Y is —C₃-C₇cycloalkyl, unsubstituted, or substituted with one or more of: -halo and-3- to 10-membered heterocycle.
 49. The method of claim 46, wherein thecompound is selected from the group consisting of:4-((1-(cyclohexylmethyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #1)

4-((1-cyclohexyl-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide (ID#2)

4-((1-cycloheptyl-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide (ID#3)

4-((1-((1-fluorocyclohexyl)methyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #4)

4-((1-(cyclopentylmethyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #5)

4-((1-(2-cyclopentylethyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #6)

4-((1-((4,4-difluorocyclohexyl)methyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #7)

4-((1-((4,4-difluorocyclohexyl)methyl)-5-fluoro-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #8)

N-hydroxy-4-((1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide(ID #9)

and4-((5-fluoro-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #10)


50. The method of claim 46, further comprising determining an expressionsignature in the subject that indicates the likely response to treatmentwith the compound, wherein the expression signature comprises expressionof the HDAC isoform.
 51. The method of claim 50, wherein the HDACisoform is selected from the group consisting of: HDAC1, HDAC3, HDAC6,and HDAC10.
 52. The method of claim 46, wherein the compound isadministered at about 10-400 mg/kg.
 53. A method of treating a histonedeacetylase (HDAC)-associated disease in a subject, comprisingadministering to the subject a compound of formula (I):

wherein: X is selected from the group consisting of: H, halo, —C₁-C₆alkyl, aryl, —C₃-C₇ cycloalkyl, and -3- to 10-membered heterocycle, anyof which is unsubstituted, or substituted with one or more of: -halo,—C₁-C₆ alkyl, —O—(C₁-C₆ alkyl), —OH, —CN, —COOR′, —OC(O)R′, NHR′,N(R′)₂, —NHC(O)R′, and —C(O)NHR′, wherein R′ is —H or —C₁-C₆ alkyl; A isselected from the group consisting of: a bond, —C₁-C₆ alkyl, and —C₃-C₇cycloalkyl, any of which is unsubstituted, or substituted with one ormore of: -halo, —C₁-C₆ alkyl, —O—(C₁-C₆ alkyl), —OH, —CN, —COOR′,—OC(O)R′, NHR′, N(R′)₂, —NHC(O)R′, and —C(O)NHR′, wherein R′ is —H or—C₁-C₆ alkyl; Y is selected from the group consisting of: H, —C₁-C₆alkyl, —C₃-C₇ cycloalkyl, aryl, and -3- to 10-membered heterocycle, anyof which is unsubstituted, or substituted with one or more of: -halo,—C₁-C₆ alkyl, —O—(C₁-C₆ alkyl), —OH, —CN, —COOR′, —OC(O)R′, NHR′,N(R′)₂, —NHC(O)R′, and —C(O)NHR′, wherein R′ is —H or —C₁-C₆ alkyl; andQ is selected from the group consisting of: —H, -halo, —C₁-C₆ alkyl,—O—(C₁-C₆ alkyl), —OH, —CN, —COOR′, —OC(O)R′, NHR′, N(R′)₂, —NHC(O)R′,and —C(O)NHR′, wherein R′ is —H or —C₁-C₆ alkyl; wherein the compoundinhibits the histone deacetylating activity of an HDAC isoform.
 54. Themethod of claim 53, wherein X is H or halo; Q is H; and A is a bond or—C₁-C₆ alkyl.
 55. The method of claim 54, wherein Y is —C₃-C₇cycloalkyl, unsubstituted, or substituted with one or more of: -halo and-3- to 10-membered heterocycle.
 56. The method of claim 53, wherein thecompound is selected from the group consisting of:4-((1-(cyclohexylmethyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #1)

4-((1-cyclohexyl-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide (ID#2)

4-((1-cycloheptyl-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide (ID#3)

4-((1-((1-fluorocyclohexyl)methyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #4)

4-((1-(cyclopentylmethyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #5)

4-((1-(2-cyclopentylethyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #6)

4-((1-((4,4-difluorocyclohexyl)methyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #7)

4-((1-((4,4-difluorocyclohexyl)methyl)-5-fluoro-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #8)

N-hydroxy-4-((1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide(ID #9)

and4-((5-fluoro-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(ID #10)


57. The method of claim 53, further comprising determining an expressionsignature in the subject that indicates the likely response to treatmentwith the compound, wherein the expression signature comprises expressionof the HDAC isoform and the HDAC isoform is selected from the groupconsisting of: HDAC1, HDAC3, HDAC6, and HDAC10.
 58. The method of claim53, wherein the HDAC-associated disease is selected from the groupconsisting of: cancer, an autoimmune disorder, an inflammatory disorder,a neurodegenerative disease, and combinations thereof.